Saccharide structures and methods of making and using such structures

ABSTRACT

Described are oligosaccharides having a protecting group at two, a plurality, a majority of, or each position in the oligosaccharide which is amenable to derivatization. Collections, libraries and methods of making and using such oligosaccharides are also described.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119 from U.S.provisional application Ser. No. 61/057,354 filed May 30, 2008, theentire contents of which is incorporated by reference.

BACKGROUND

Polysaccharides such as heparin, heparan sulfate, chondroitin sulfate,dermatan sulfate and hyaluronic acid are complex and heterogeneousmixtures of saccharide structures. Their biological properties andtherapeutic applications are a reflection of this complexity.

SUMMARY

The disclosure is based, in part, on the discovery of saccharidescaffolds that can be used to provide oligosaccharides, e.g., anoligosaccharide having a preselected sequence, as well as substantiallyhomogenous or defined mixtures of oligosaccharides.

Accordingly, in one aspect, the disclosure features an oligosaccharidethat is a disaccharide or larger. The oligosaccharide can have apreselected sequence, e.g., a sequence of saccharide structures having apreselected pattern of derivatization. The oligosaccharide allows thedesign and synthesis of oligosaccharide structures having preselectedcomplex patterns of derivatization, e.g., preselected complex patternsof sulfation or acetylation. In some embodiments, the oligosaccharidehas only two different protecting groups. In embodiments the twoprotecting groups have different reactivities. One protecting group isreplaced to a first degree, e.g., substantially completely replaced,with a derivatizing group under selected conditions. The otherprotecting group gives relatively less, e.g., it gives substantially noderivatization, under the same conditions. The oligosaccharide can be,e.g., a disaccharide, a trisaccharide, a tetrasaccharide, apentasaccharide, a hexasaccharide, an octasaccharide, a nonasaccharide,a decasaccharide, a dodecasaccharide, tetradecasaccharide,hexadecasaccharide, or octadecasaccharide. (Saccharides of the inventioncan have an even or odd number of monosaccharide subunits.)

In one embodiment, the disclosure features an oligosaccharide comprisinga plurality of disaccharide units, wherein one, two, a plurality, amajority of, or each position amenable to sulfation or acetylationwithin a disaccharide unit in the plurality is protected either with afirst protecting group that allows derivatization, e.g., sulfation oracetylation, or a second protecting group that does not allowderivatization, e.g., sulfation or acetylation, wherein the identity ofeach protecting group of each disaccharide unit is independent of theidentity of any other protecting group in the disaccharide unit, andwherein the identity of each disaccharide unit is independent of theidentity of the other disaccharide units within the oligosaccharide.

In one embodiment at least one, two, a plurality, a majority, at least50, 60, 70, 80, or 90%, or all of the disaccharide units of theplurality, or in the oligosaccharide, have each (or a) position amenableto derivatization protected with one of the two protecting groups. In anembodiment at least one, two, a plurality, a majority, at least 50, 60,70, 80, or 90%, or all of the disaccharide units of the plurality or inthe oligosaccharide have each position amenable to derivatizationprotected with one of the two protecting groups and in a furtherembodiment each disaccharide of the plurality or in the oligosaccharidehas includes at least one of both protecting groups.

In one embodiment, the disaccharide unit or units within theoligosaccharide are protected with a protecting group that allowsderivatization and the protecting group that allows derivatization isthe same protecting group at each position. In one embodiment, thedisaccharide unit or units are protected with a protecting group thatdoes not allow derivatization and the protecting group that does notallow derivatization is the same protecting group at each position.

In one embodiment, within all of the disaccharide units of theoligosaccharide, each position amenable to derivatization, e.g.,sulfation or acetylation, is protected with either a protecting groupthat allows derivatization, e.g., sulfation or acetylation, or aprotecting group that does not allow derivatization, e.g., sulfation oracetylation. In one embodiment, each position protected with aprotecting group that allows derivatization is protected with the sameprotecting group and each position protected with a protecting groupthat does not allow derivatization is protected with the same protectinggroup.

In one embodiment, the protecting group can be a hydroxyl protectinggroup such as, e.g., silyl ethers, ethyl ethers, substituted benzylethers and esters. In some embodiments, the protecting group can be anamine protecting group such as, e.g., carbamates and substitutedcarbamates. In one embodiment, the protecting group that allowsderivatization is selected from levulinoyl, benzyl (Bn), benzoyl (Bz),methoxybenzyl (MPM), azide, allyl and silyl ether protecting group(e.g., tBDMS or tBDPS) and the protecting group that does not allowderivatization is selected from levulinoyl, benzyl, benzoyl, MPM, azide,allyl and silyl ether protecting group (e.g., tBDMS or tBDPS), so longas the protecting group that allows derivatization and the protectinggroup that does not allow derivatization are orthogonal protectinggroups. In one embodiment, the protecting group that allowsderivatization is a benzoyl and/or a benzoyl containing group and theprotecting group that does not allow derivatization is a benzyl, abenzyl containing group and/or an azide. In another embodiment, theprotecting group that allows derivatization is a benzyl and/or a benzylcontaining group and the protecting group that does not allowderivatization is a benzoyl, a benzoyl containing group and/or an azide.In one embodiment, the protecting group that allows derivatization is alevulinoyl and the protecting group that does not allow derivatizationis a benzoyl, a benzoyl containing group and/or an azide. In anotherembodiment, the protecting group that allows derivatization is a benzoyland/or a benzoyl containing group and the protecting group that does notallow derivatization is a levulinoyl and/or an azide.

In one embodiment, the disaccharide unit or units are an uronic acid(e.g., iduronic acid and/or glucuronic acid) and a hexosamine (e.g.,glucosamine and galactosamine). In one embodiment, the disaccharide unitor units are N-acetylgalactosamine or N-acetylglucosamine and an uronicacid (e.g., glucuronic acid and/or iduronic acid).

In one embodiment, the oligosaccharide comprises a disaccharide havingthe structure of:

wherein R₈ is an alkyl group, e.g., ethyl, methyl, propyl, butyl,pentyl, etc. group, and wherein R₁, R₂, R₅, R₆ and R₇ are either a firstprotecting group that allows derivatization, e.g., sulfation oracetylation, or a second protecting group that does not allowderivatization, e.g., sulfation or acetylation. In one embodiment, ateach position within the oligosaccharide having the protecting groupthat allows derivatization, the protecting group is the same protectinggroup for protecting groups that allow derivatization. In oneembodiment, at each position within the oligosaccharide having aprotecting group that does not allow derivatization, the protectinggroup is the same protecting group that does not allow derivatization.In one embodiment, at each position within the oligosaccharide havingthe protecting group that allows derivatization, the protecting group isthe same protecting group and at each position within theoligosaccharide having a protecting group that does not allowderivatization, the protecting group is the same protecting group.

In one embodiment, the oligosaccharide is a decasaccharide thatcomprises, e.g., consists essentially of:

wherein each of X1, X2, X3 and X4 is independently A or B, and wherein

and wherein R₈ for each occurrence of A or B is an alkyl group, e.g., anethyl, methyl, propyl, butyl, pentyl, etc. group and wherein R₁, R₂, R₅,R₆, and R₇ for each occurrence of A or B is a protecting group selectedfrom either a first protecting group that allows derivatization, e.g.,sulfation or acetylation, or a second protecting group that does notallow derivatization, e.g., sulfation or acetylation. In one embodiment,at each position within the oligosaccharide having the protecting groupthat allows derivatization, the protecting group is the same protectinggroup. In one embodiment, at each position within the decasaccharidehaving a protecting group that does not allow derivatization, theprotecting group is the same protecting group. In one embodiment, ateach position within the decasaccharide having the protecting group thatallows derivatization, the protecting group is the same protecting groupand at each position within the decasaccharide having a protecting groupthat does not allow derivatization, the protecting group is the sameprotecting group. In embodiments, the selection of one or more of R₁,R₂, R₅, R₆, and R₇ can differ between a first and second group A, an Aand B, or a first and second B.

In one embodiment, the oligosaccharide is a decasaccharide thatcomprises, e.g., consists essentially of:

wherein each of X5, X6, X7 and X8 is independently C or D, and wherein

and wherein R₈ for each occurrence of C or D is an alkyl group, e.g., anethyl, methyl, propyl, butyl, pentyl, etc. group and wherein R₁, R₂, R₅,R₆, and R₇ for each occurrence of C or D is a protecting group selectedfrom either a first protecting group that allows derivatization, e.g.,sulfation or acetylation, or a second protecting group that does notallow derivatization, e.g., sulfation or acetylation. In one embodiment,at each position within the oligosaccharide having the protecting groupthat allows derivatization, the protecting group is the same protectinggroup. In one embodiment, at each position within the decasaccharidehaving a protecting group that does not allow derivatization, theprotecting group is the same protecting group. In one embodiment, ateach position within the decasaccharide having the protecting group thatallows derivatization, the protecting group is the same protecting groupand at each position within the decasaccharide having a protecting groupthat does not allow derivatization, the protecting group is the sameprotecting group. In embodiments, the selection of one or more of R₁,R₂, R₅, R₆, and R₇ can differ between a first and second group C, a Cand D, or a first and second D.

The disclosure also features an oligosaccharide, e.g., anoligosaccharide described above, wherein at any position described aboveas having a protecting group that allows derivatization, there is asulfate group, and the remaining positions have a protecting group thatdoes not allow derivatization.

The disclosure also features an oligosaccharide, e.g., anoligosaccharide described above, wherein at any position described aboveas having a protecting group that does not allow derivatization, thereis one or more hydrogen, and the remaining positions have a protectinggroup that allows derivatization.

In one aspect, the disclosure features a disaccharide having aprotecting group at two, a plurality, a majority of, or each position inthe disaccharide amenable to derivatization. The protecting group at anygiven position can be a first protecting group that allowsderivatization, e.g., sulfation or acetylation, or a second protectinggroup that does not allow derivatization, e.g., sulfation oracetylation. As described elsewhere herein, these are useful, forproviding oligosaccharides, or libraries thereof, having preselectedsequences and/or levels or patterns of derivatization, e.g., sulfationor acetylation.

In one embodiment, the disclosure features a protected disaccharidehaving a protecting group at each position amenable to sulfation oracetylation within the disaccharide, wherein the protecting group ateach position is either a first protecting group that allowsderivatization, e.g., sulfation or acetylation, or a second protectinggroup that does not allow derivatization, e.g., sulfation oracetylation, and, e.g., the identity of each protecting group isindependent of the identity of a protecting group at any other positionin the saccharide structure.

In one embodiment, the disaccharide has a preselected pattern ofprotecting groups, e.g., which when derivatized will provide apreselected pattern of derivatization, e.g., sulfation or acetylation.

In an embodiment each position amenable to derivatization is protectedwith one of the two protecting groups and in an embodiment at least oneof each group is present in the disaccharide.

In one embodiment, the protecting group can be a hydroxyl protectinggroup such as, e.g., silyl ethers, ethyl ethers, substituted benzylethers and esters. In some embodiments, the protecting group can be anamine protecting group such as, e.g., carbamates and substitutedcarbamates. In one embodiment, the protecting group that allowsderivatization is selected from levulinoyl, benzyl (Bn), benzoyl (Bz),methoxybenzyl (MPM), azide, allyl and silyl ether protecting group(e.g., tBDMS or tBDPS), and the protecting group that does not allowderivatization is selected from levulinoyl, benzyl, benzoyl, MPM, azide,allyl and silyl ether protecting group (e.g., tBDMS or tBDPS), so longas the protecting group that allows derivatization and the protectinggroup that does not allow derivatization are orthogonal protectinggroups. In one embodiment, the protecting group that allowsderivatization is a benzoyl and/or a benzoyl containing group and theprotecting group that does not allow derivatization is a benzyl, abenzyl containing group and/or an azide. In another embodiment, theprotecting group that allows derivatization is a benzyl and/or a benzylcontaining group and the protecting group that does not allowderivatization is a benzoyl, a benzoyl containing group and/or an azide.In one embodiment, the protecting group that allows derivatization is alevulinoyl and the protecting group that does not allow derivatizationis a benzoyl, a benzoyl containing group and/or an azide. In anotherembodiment, the protecting group that allows derivatization is a benzoyland/or a benzoyl containing group and the protecting group that does notallow derivatization is levulinoyl and/or an azide.

In one embodiment, the disaccharide is an uronic acid (e.g., iduronicacid or glucuronic acid) and a hexosamine (e.g., glucosamine,galactosamine). In one embodiment, the disaccharide isN-acetylgalactosamine or N-acetylglucosamine and an uronic acid (e.g.,glucuronic acid and/or iduronic acid).

In one embodiment, at least one position within the disaccharide thatcan form linkages with another saccharide structure is protected with aprotecting group. Examples of protecting groups that can be at positionswithin the disaccharide that are involved with attaching thedisaccharide to another saccharide structure can be any orthogonalhydroxyl protecting groups from, e.g., ethers, substituted ethers, silylethers, acetals, esters, etc. Exemplary protecting groups include, butare not limited to, levulinoyl, benzoyl, tert-butyldimethylsilyl(tBDMS), tert-butyldiphenylsilyl (tBDPS), 2-Naphthyl (2-NAP) and9-Fluorenylmethoxycarbonyl (Fmoc).

In one embodiment, the disaccharide has one of the following structuresI, II, III or IV:

wherein R₁, R₂, R₅, R₆ and R₇ are protecting groups selected from eithera first protecting group that allows derivatization, e.g., sulfation oracetylation, or a second protecting group that does not allowderivatization, wherein the identity of the protecting group at any ofR₁, R₂, R₅, R₆ and R₇ is independent of the identity of a protectinggroup at any of the other positions; and wherein R₈ is a hydrogen or analkyl group, e.g., an ethyl, methyl, propyl, butyl, pentyl, etc. In oneembodiment, the protecting group that allows derivatization is a benzyl,a benzyl containing group and the protecting group that does not allowderivatization is a benzoyl, a benzoyl containing group and/or an azide.In one embodiment, the protecting group that allows derivatization is abenzoyl and/or a benzoyl containing group and the protecting group thatdoes not allow derivatization is a benzyl, a benzyl containing groupand/or an azide.

In one embodiment, R₃ is a protecting group, e.g., a protecting groupdescribed herein, e.g., a levulinoyl, that can be at a position withinthe monosaccharide that is involved with attaching the monosaccharide toanother saccharide structure.

In one embodiment, R₄ is a protecting group, e.g., a protecting groupdescribed herein, e.g., a benzoyl or a 2Nap, that can be at a positionwithin the monosaccharide that is involved with attaching themonosaccharide to another saccharide structure.

In one embodiment, the disaccharide is any one of the disaccharidesdescribed in Table I, Table II, FIG. 6, FIG. 7, FIG. 14 or FIG. 15.

The disaccharides can be incorporated into larger oligosaccharides,e.g., a trisaccharide, a tetrasaccharide, a pentasaccharide, ahexasaccharide, an octasaccharide, a decasaccharide, a dodecasaccharide,tetradecasaccharide, hexadecasaccharide, or octadecasaccharide.

In another aspect, the disclosure features a monosaccharide having aprotecting group at two, a plurality of, a majority of, or each positionamenable to derivatization within the monosaccharide. The protectinggroup at any given position can be a first protecting group that allowsderivatization, e.g., sulfation or acetylation, or a second protectinggroup that does not allow derivatization, e.g., sulfation oracetylation. The identity of each protecting group each at position isindependent of the protecting group at any other position. As describedelsewhere herein, these are useful, for providing disaccharides orlarger oligosaccharides, or libraries thereof, having preselectedsequences and/or levels or patterns of derivatization, e.g., sulfationor acetylation.

In an embodiment each position amenable to derivatization is protectedwith one of the two protecting groups and in an embodiment at least oneof each group is present.

In one embodiment, the disclosure features a protected monosaccharidehaving a protecting group at each position within the monosaccharideamenable to sulfation or acetylation, wherein the protecting group ateach position is either a first protecting group that allowsderivatization, e.g., sulfation or acetylation, or a second protectinggroup that does not allow derivatization, e.g., sulfation oracetylation, and the identity of a protecting group at each positionamendable to derivatization is independent of the identity of aprotecting group at any other position amendable to derivatization inthe monosaccharide structure.

In one embodiment, the protecting group can be a hydroxyl protectinggroup such as, e.g., silyl ethers, ethyl ethers, substituted benzylethers and esters. In some embodiments, the protecting group can be anamine protecting group such as, e.g., carbamates and substitutedcarbamates. In one embodiment, the protecting group that allowsderivatization is selected from levulinoyl, benzyl (Bn), benzoyl (Bz),methoxybenzyl (MPM), azide, allyl and silyl ether protecting group(e.g., tBDMS or tBDPS), and the protecting group that does not allowderivatization is selected from levulinoyl, benzyl, benzoyl, MPM, azide,allyl and silyl ether protecting group (e.g., tBDMS or tBDPS), so longas the protecting group that allows derivatization and the protectinggroup that does not allow derivatization are orthogonal protectinggroups. In one embodiment, the protecting group that allowsderivatization is a benzoyl and/or a benzoyl containing group and theprotecting group that does not allow derivatization is a benzyl, abenzyl containing group and/or an azide. In another embodiment, theprotecting group that allows derivatization is a benzyl and/or a benzylcontaining group and the protecting group that does not allowderivatization is a benzoyl, a benzoyl containing group and/or an azide.In one embodiment, the protecting group that allows derivatization is alevulinoyl and the protecting group that does not allow derivatizationis a benzoyl, a benzoyl containing group and/or an azide. In anotherembodiment, the protecting group that allows derivatization is a benzoyland/or a benzoyl containing group and the protecting group that does notallow derivatization is levulinoyl and/or an azide.

In one embodiment, the monosaccharide is an uronic acid (e.g., iduronicacid or glucuronic acid) or a hexosamine (e.g., glucosamine,galactosamine). In one embodiment, the monosaccharide isN-acetylgalactosamine, N-acetylglucosamine or an uronic acid (e.g.,glucuronic acid and/or iduronic acid).

In one embodiment, at least one position within the monosaccharide thatcan form linkages with another saccharide structure is protecting with aprotecting group. Examples of protecting groups that can be at positionswithin the monosaccharide that are involved with attaching themonosaccharide to another saccharide structure can be any orthogonalhydroxyl protecting groups from, e.g., ethers, substituted ethers, silylethers, acetals, esters, etc. Exemplary protecting groups include, butare not limited to, levulinoyl, benzoyl, tert-butyldimethylsilyl(tBDMS), tert-butyldiphenylsilyl (tBDPS), 2-NaphthalenesulphonylL-aspartyl-(2-phenethyl)amide (2-NAP) and Fmoc.

In one embodiment, the monosaccharide has one of the followingstructures:

wherein R₈ is a hydrogen or an alkyl group, e.g., an ethyl, methyl,propyl, butyl, pentyl, etc.; and wherein R₁ and R₂ are protecting groupsselected from either a first protecting group that allowsderivatization, e.g., sulfation or acetylation, or a second protectinggroup that does not allow derivatization, wherein the identity of theprotecting group at R₁ and R₂ is independent of the protecting group atthe other position. In some embodiments, R₁ and R₂ both have aprotecting group that allows derivatization; R₁ and R₂ both have aprotecting group that does not allow derivatization, R₁ has a protectinggroup that allows derivatization and R₂ protecting group that does notallow derivatization; or R₂ has a protecting group that allowsderivatization and R₁ has a protecting group that does not allowderivatization. In one embodiment, the protecting group that allowsderivatization is a benzyl and the protecting group that does not allowderivatization is a benzoyl. In one embodiment, the protecting groupthat allows derivatization is a benzoyl and the protecting group thatdoes not allow derivatization is a benzyl.

In one embodiment, R₉ is a protecting group, e.g., a protecting groupdescribed herein, e.g., a levulinoyl or a tBDMS, that can be at aposition within the monosaccharide that can attach the monosaccharide toanother saccharide structure.

In one embodiment, the monosaccharide has one of the followingstructures:

wherein R₅, R₆ and R₇ are protecting groups selected from either a firstprotecting group that allows derivatization, e.g., sulfation oracetylation, or a second protecting group that does not allowderivatization, wherein the identity of the protecting group at R₅, R₆and R₇ is independent of the identity of the protecting group at theother positions in the monosaccharide. In some embodiments, R₅, R₆ andR₇ all have a protecting group that allows derivatization; R₅, R₆ and R₇all have a protecting group that does not allow derivatization, R₅ andR₆ have a protecting group that allows derivatization and R₇ has aprotecting group that does not allow derivatization; R₅ has a protectinggroup that allows derivatization and R₆ and R₇ have a protecting groupthat does not allow derivatization; R₅ and R₇ have a protecting groupthat allows derivatization and R₆ has a protecting group that does notallow derivatization; R₅ and R₆ have a protecting group that does notallow derivatization and R₇ has a protecting group that allowsderivatization; R₅ has a protecting group that does not allowderivatization and R₆ and R₇ have a protecting group that allowsderivatization; R₅ and R₇ have a protecting group that does not allowderivatization and R₆ has a protecting group that allows derivatization.In one embodiment, the protecting group that allows derivatization is abenzyl and/or a benzyl containing group and the protecting group thatdoes not allow derivatization is a benzoyl, a benzoyl containing groupand/or an azide. In one embodiment, the protecting group that allowsderivatization is a benzoyl and/or a benzyl containing group and theprotecting group that does not allow derivatization is a benzyl, abenzyl containing group and/or an azide.

In one embodiment, R₁₀ is a protecting group, e.g., a protecting groupdescribed herein, e.g., a benzoyl or a 2Nap, that can be at a positionwithin the monosaccharide that is involved with attaching themonosaccharide to another saccharide structure.

In one embodiment, the monosaccharide is a monosaccharide provided inFIG. 1-5, 12 or 13.

In another aspect, the disclosure features a method of making anoligosaccharide that is a disaccharide or larger, e.g., a sequence ofsaccharide structures having a preselected pattern of derivatization.Embodiments of the method allow the design and synthesis ofoligosaccharide structures having preselected complex patterns ofderivatization, e.g., preselected complex patterns of sulfation oracetylation. Saccharide structures or subunits, each having theappropriate pattern of protecting groups, are joined together to allowthe production of the larger saccharide structure having the preselectedpattern of derivatization. A single derivatizing reaction can thenprovide the preselected pattern of derivatization. Embodiments of themethod accomplish this with the use of only two different protectinggroups. In an embodiment each of the two protecting groups havedifferent reactivity. One protecting group is replaced to a firstdegree, e.g., substantially completely replaced, with a derivatizinggroup under selected conditions. The other protecting group givesrelatively less, e.g., it gives substantially no derivatization, underthe same conditions. Embodiments rely on a library of different subunitsor saccharide structures. The library provides a plurality ofoligosaccharide structures having diverse patterns of the two protectinggroups. Thus, one can select a first library member having a pattern ofprotecting groups which, upon derivatization can give a selected patternof derivatization. As referred to above, the first library member isjoined to one or more subsequent library members having selectedpatterns of protecting groups and selected to provide a pattern ofprotecting groups. As referred to above, a single reaction can be usedto derivatize the entire larger saccharide to provide theoligosaccharide having a preselected pattern of derivatization. Theoligosaccharide can be, e.g., a disaccharide, a trisaccharide, atetrasaccharide, a pentasaccharide, a hexasaccharide, an octasaccharide,a decasaccharide, a dodecasaccharide, tetradecasaccharide,hexadecasaccharide, or octadecasaccharide.

The method includes:

providing a first protected saccharide structure, wherein the saccharideis a monosaccharide or larger, and wherein one, two, a plurality of, amajority of, or all positions in the saccharide structure amenable toderivatization, e.g., sulfation or acetylation, are protected witheither a protecting group that allows derivatization, e.g., sulfation oracetylation, or a protecting group that does not allow derivatization,e.g., sulfation or acetylation, and wherein the identity of a protectinggroup at each position is independent of the identity of a protectinggroup at any other position in the saccharide structure;

providing a second saccharide structure, wherein the saccharide is amonosaccharide or larger, and optionally, wherein one, two, a pluralityof, a majority of, or all positions in the saccharide structure amenableto derivatization, e.g., sulfation or acetylation, are protected witheither a protecting group that allows derivatization, e.g., sulfation oracetylation, or a protecting group that does not allow derivatization,e.g., sulfation or acetylation, and wherein the identity of a protectinggroup at each position is independent of the identity of a protectinggroup at any other position in the saccharide structure; and

attaching the first saccharide structure to the second saccharidestructure, to thereby make an oligosaccharide of preselected sequence.

In one embodiment at least one, two, a plurality, a majority, at least50, 60, 70, 80, or 90%, or all of the protected saccharide units haveeach (or a) position amenable to derivatization protected with one ofthe two protecting groups. In an embodiment at least one, two, aplurality, a majority, at least 50, 60, 70, 80, or 90%, or all of theprotected saccharide units have each position amenable to derivatizationprotected with one of the two protecting groups and in a furtherembodiment each protected saccharide of the includes at least one ofboth protecting groups.

In one embodiment, the method includes making an oligosaccharide with asequence having a preselected pattern of derivatization, e.g., apreselected pattern of sulfation or acetylation, e.g., a sequence havinga first saccharide structure having a first pattern of derivatization,e.g., a sulfate derivatized at position R₁ of an uronic acid, and asecond saccharide structure having a second pattern of derivatization,e.g., a sulfate at position R₆ of a hexosamine. The method includes:

providing a first protected saccharide structure which when derivatizedwill provide a saccharide structure having a first preselected patternof derivatization, wherein the saccharide is a monosaccharide or larger,and wherein all positions in the saccharide structure amenable toderivatization, e.g., sulfation or acetylation, are protected witheither a protecting group that allows derivatization, e.g., sulfation oracetylation, or a protecting group that does not allow derivatization,e.g., sulfation or acetylation;

providing a second saccharide structure which when derivatized willprovide a saccharide structure having a second preselected pattern ofderivatization, wherein the saccharide is a monosaccharide or larger,wherein all positions in the saccharide structure amenable toderivatization, e.g., sulfation or acetylation, are protected witheither a protecting group that allows derivatization, e.g., sulfation oracetylation, or a protecting group that does not allow derivatization,e.g., sulfation or acetylation; and attaching the first saccharidestructure to the second saccharide structure, to thereby make theoligosaccharide with a sequence having a preselected pattern ofderivatization.

In one embodiment, positions in the second saccharide structure that areamenable to derivatization, e.g., sulfation or acetylation, areprotected with either a protecting group that allows derivatization,e.g., sulfation or acetylation, or a protecting group that does notallow derivatization, e.g., sulfation or acetylation, and the identityof a protecting group at each position amendable to derivatization isindependent of the identity of a protecting group at any other positionamendable to derivatization in the saccharide structure.

In one embodiment, if more than one position in the first saccharidestructure is protected with a protecting group that allowsderivatization, the protecting group that allows derivatization is thesame protecting group at each position and/or if more than one positionin the first saccharide structure is protected with a protecting groupthat does not allow derivatization, the protecting group that does notallow derivatization is the same protecting group at each position.

In one embodiment, if more than one position in the second saccharidestructure is protected with a protecting group that allowsderivatization, the protecting group that allows derivatization is thesame protecting group at each position and/or if more than one positionin the second saccharide structure is protected with a protecting groupthat does not allow derivatization, the protecting group that does notallow derivatization is the same protecting group at each position.

In one embodiment, at every position within the oligosaccharideprotected with a protecting group that allows derivatization, theprotecting group is the same protecting group at each position. In oneembodiment, at every position within the oligosaccharide protected witha protecting group that does not allow derivatization, the protectinggroup is the same protecting group at each position. In one embodiment,at every position within the oligosaccharide protected with a protectinggroup that allows derivatization, the protecting group is the sameprotecting group at each position and at every position within theoligosaccharide protected with a protecting group that does not allowderivatization, the protecting group is the same protecting group ateach position.

In one embodiment, the protecting group can be a hydroxyl protectinggroup such as, e.g., silyl ethers, ethyl ethers, substituted benzylethers and esters. In some embodiments, the protecting group can be anamine protecting group such as, e.g., carbamates and substitutedcarbamates. In one embodiment, the protecting group that allowsderivatization is selected from levulinoyl, benzyl (Bn), benzoyl (Bz),methoxybenzyl (MPM), azide, allyl and silyl ether protecting group(e.g., tBDMS or tBDPS), and the protecting group that does not allowderivatization is selected from levulinoyl, benzyl, benzoyl, MPM, azide,allyl and silyl ether protecting group (e.g., tBDMS or tBDPS), so longas the protecting group that allows derivatization and the protectinggroup that does not allow derivatization are orthogonal protectinggroups. In one embodiment, the protecting group that allowsderivatization is a benzoyl and/or a benzoyl containing group and theprotecting group that does not allow derivatization is a benzyl, abenzyl containing group and/or an azide. In another embodiment, theprotecting group that allows derivatization is a benzyl and/or a benzylcontaining group and the protecting group that does not allowderivatization is a benzoyl, a benzoyl containing group and/or an azide.In one embodiment, the protecting group that allows derivatization is alevulinoyl and the protecting group that does not allow derivatizationis a benzoyl, a benzoyl containing group and/or an azide. In anotherembodiment, the protecting group that allows derivatization is a benzoyland/or a benzoyl containing group and the protecting group that does notallow derivatization is levulinoyl and/or an azide.

In one embodiment, the method further includes providing a thirdsaccharide structure, wherein the third saccharide structure is amonosaccharide or larger; and attaching the third saccharide structureto the saccharide structure formed from the first and second saccharidestructure. In one embodiment, the third saccharide structure has aprotecting group at all positions in the saccharide structure amenableto derivatization, e.g., sulfation or acetylation, wherein theprotecting group is either a protecting group that allowsderivatization, e.g., sulfation or acetylation, or a protecting groupthat does not allow derivatization, e.g., sulfation or acetylation, andthe identity of a protecting group at each position amendable toderivatization is independent of the identity of a protecting group atany other position amendable to derivatization in the first, second orthird saccharide structure. In one embodiment, the method can furtherinclude providing and attaching a fourth, fifth, sixth, seventh, etc.saccharide structure to make the oligosaccharide. Any of the fourth,fifth, sixth, seventh, etc. saccharide structures can be, e.g., asaccharide structure protected at every position amenable toderivatization, e.g., sulfation or acetylation, with either a protectinggroup that allows derivatization, e.g., sulfation or acetylation, or aprotecting group that does not allow derivatization, e.g., sulfation oracetylation, and the identity of the protecting group at each positionamendable to derivatization is independent of the identity of theprotecting group at any other position amendable to derivatization inany of the other saccharide structures.

In one embodiment, the saccharide structure (e.g., the first, second,third, fourth, etc. saccharide structure) is a monosaccharide, e.g., amonosaccharide described herein, a disaccharide, e.g., a disaccharidedescribed herein, a tetrasaccharide, a pentasaccharide, ahexasaccharide, an octasaccharide, or a decasaccharide. In oneembodiment, the first saccharide structure is a disaccharide, e.g., adisaccharide described herein, and the second saccharide structure isselected from a monosaccharide, a disaccharide, a trisaccharide, atetrasaccharide, a pentasaccharide, a hexasaccharide, a heptasaccharide,and an octasaccharide.

In one embodiment, the method includes providing a first disaccharidestructure, e.g., a disaccharide described herein; attaching a seconddisaccharide structure, e.g., a disaccharide structure described herein,to the first disaccharide structure to provide a first tetrasaccharidestructure; providing a third disaccharide structure, e.g., adisaccharide described herein; attaching a fourth disaccharidestructure, e.g., a disaccharide structure described herein, to the thirddisaccharide structure to provide a second tetrasaccharide structure;attaching the first tetrasaccharide structure to the secondtetrasaccharide structure to provide an octasaccharide structure; andattaching a fifth disaccharide structure, e.g., a disaccharide structuredescribed herein, to the octasaccharide structure to thereby provide adecasaccharide structure.

In one embodiment, at least one position within the first saccharidestructure that attaches the first saccharide structure to the secondsaccharide structure is protected with a protecting group. In oneembodiment, at least one position within the second saccharide structurethat attaches the second saccharide structure to the first saccharidestructure is protected with a protecting group. In one embodiment, if athird saccharide structure is attached to the second saccharidestructure, the second saccharide structure has a protecting group at aposition within the second saccharide structure that attaches to thefirst saccharide structure and a protecting group at a position withinthe second saccharide structure that attaches to the third saccharidestructure. When the second saccharide structure is attached to a thirdsaccharide structure, the third saccharide structure, preferably, has aprotecting group at a position within the third saccharide structurethat attaches with the second saccharide structure. In one embodiment,if a fourth saccharide structure is attached to the third saccharidestructure, the third saccharide structure has a protecting group at aposition within the third saccharide structure that attaches to thesecond saccharide structure and a protecting group at a position withinthe third saccharide structure that attaches to the fourth saccharidestructure. When the third saccharide structure is attached to a fourthsaccharide structure, the fourth saccharide structure, preferably, has aprotecting group the position within the fourth saccharide structurethat attaches with the third saccharide structure. Preferably everysaccharide structure used in the described methods has a protectinggroup at every position in the saccharide structure that attaches onesaccharide structure to another saccharide structure. Examples ofprotecting groups that can be at positions within the saccharidestructures that are involved with attaching one saccharide structure toanother can be any orthogonal hydroxyl protecting groups from, e.g.,ethers, substituted ethers, silyl ethers, acetals, esters, etc.Exemplary protecting groups include, but are not limited to, levulinoyl,benzoyl, tert-butyldimethylsilyl (tBDMS), tert-butyldiphenylsilyl(tBDPS), 2-Naphthalenesulphonyl L-aspartyl-(2-phenethyl)amide (2-NAP)and Fmoc.

In one embodiment, the first saccharide structure includes an uronicacid (e.g., a glucuronic acid or iduronic acid), a hexosamine (e.g., aglucosamine), or a combination or combinations of an uronic acid (e.g.,a glucuronic acid or iduronic acid), and a hexosamine (e.g., aglucosamine). In one embodiment, the oligosaccharide comprises uronicacid (e.g., a glucuronic acid and/or iduronic acid) and hexosamine(e.g., glucosamine) and the positions amendable to derivatization arethe positions amenable to sulfation or acetylation in heparin or heparansulfate. In another embodiment, the first saccharide structure includesan N-acetylgalactosamine, an uronic acid (e.g., a glucuronic acid oriduronic acid), or a combination or combinations of anN-acetylgalactosamine and an uronic acid (e.g., a glucuronic acid oriduronic acid). In one embodiment, the oligosaccharide comprises anN-acetylgalactosamine and an uronic acid (e.g., a glucuronic acid oriduronic acid) and the positions amenable to derivatization arepositions amenable to sulfation or acetylation in chondroitin sulfate.In one embodiment, the oligosaccharide comprises anN-acetylgalactosamine and an uronic acid (e.g., a glucuronic acid oriduronic acid) and the positions amenable to derivatization arepositions amenable to sulfation or acetylation in dermatan sulfate. Inone embodiment, the oligosaccharide comprises an N-acetylglucosamine andan uronic acid (e.g., a glucuronic acid or iduronic acid) and thepositions amenable to derivatization are positions amenable to sulfationor acetylation in hyaluronic acid.

In one embodiment, the saccharide structure, e.g., the first, second,third, fourth, etc. saccharide structure is a monosaccharide, adisaccharide or an oligosaccharide larger than a disaccharide asdescribed herein.

In one embodiment, the method further includes deprotecting one or morepositions within the saccharide structure or saccharide structures thatattaches one saccharide structure to another to form an unprotectedmoiety or moieties. The deprotected moiety or moieties can then be usedto attach one saccharide structure to another. In one embodiment, onesaccharide structure, e.g., the first saccharide structure, is attachedto another saccharide structure, e.g., the second saccharide structure,using a reaction mixture that comprises a catalyst, e.g., TMSOTf orTESOTf.

In one embodiment, the oligosaccharide being made is an oligosaccharidedescribed herein.

In an embodiment, the method is repeated to form a collection or libraryof oligosaccharides.

In one embodiment, the method further comprises deprotecting theprotecting group that allows derivatization, e.g., sulfation oracetylation, to form an unprotected moiety or moieties. The method canfurther comprise forming derivative moieties, e.g., sulfate or acetate,at the deprotected position or positions.

In one embodiment, the method includes deprotecting a protecting groupthat is not amendable to derivatization to form an unprotected moiety ormoieties. The method can further include adding hydrogen at thedeprotected position or positions not amendable to derivatization.

In an embodiment, the method is repeated to form a collection or libraryof oligosaccharides having preselected sequences.

In one aspect, the disclosure features a method of making anoligosaccharide structure having a defined level or pattern ofderivatization, e.g., sulfation or acetylation, comprising:

providing an oligosaccharide structure, e.g., an oligosaccharidestructure having positions in the oligosaccharide structure amenable toderivatization, e.g., sulfation or acetylation, protected with either aprotecting group that allows derivatization, e.g., sulfation oracetylation, or a protecting group that does not allow derivatization,e.g., as described herein;

deprotecting a class of protected positions in the oligosaccharide,e.g., deprotecting positions in the oligosaccharide that have protectinggroups that allow derivatization, to form unprotected moieties andforming derivatized moieties, e.g., sulfate moieties and acetatemoieties, on the deprotected positions.

In one embodiment the method includes:

deprotecting a subsequent second class of protected positions in theoligosaccharide, e.g., deprotecting positions in the oligosaccharidethat have protecting groups that do not allow derivatization, to formunprotected moieties and forming hydrogen moieties on the second classof deprotected positions.

In one embodiment the method is repeated to form a library ofoligosaccharides having preselected levels of patterns of substituents,e.g., sulfation, acetylation.

Although deprotection is sometimes referred to separately, it isunderstood that deprotection and derivatization can occur sequentiallyor simultaneously. In addition, it is understood that forming aderivatized moiety and a hydrogen moiety can occur sequentially orsimultaneously.

In one embodiment, the oligosaccharide is an oligosaccharide describedherein.

In one aspect, the disclosure features a disaccharide having diverseprotecting groups (diversely protected saccharides). The disaccharidescan be incorporated into larger structures, e.g., a trisaccharide, atetrasaccharide, a pentasaccharide, a hexasaccharide, an octasaccharide,a decasaccharide, a dodecasaccharide, tetradecasaccharide,hexadecasaccharide, or octadecasaccharide. These are useful, e.g., forproviding oligosaccharides, or libraries thereof, having preselectedsequences and/or levels or patterns of derivatization, e.g., sulfationor acetylation.

In one embodiment, the disaccharide has one of the following structuresI, II, III or IV:

wherein R₈ is an alkyl group, e.g., an ethyl, methyl, propyl, butyl,pentyl, etc. group, and wherein each of R₁, R₂, R₅, R₆, and R₇ aredistinct from one another or a subset of R₁, R₂, R₅, R₆, and R₇ aredistinct from one another, e.g., three or more of these positions aredistinct from each other. In one embodiment, R₁, R₂, R₅, R₆, and R₇, areorthogonal protecting groups and thus, each protecting group, R₁, R₂,R₅, R₆, and R₇, is selected such that any one can be individuallyremoved, without removing the others, to allow reaction of the protectedposition with another moiety, e.g., to result in the placement of asubstituent, e.g., a sulfate or acetate, at the protected moiety. Forexample, the protecting group at R₂ can be removed without removing R₁,R₅, R₆, and R₇.

In one embodiment, the protecting group at any of R₁, R₂, and R₆, and R₇can be a hydroxyl protecting group such as, e.g., silyl ethers, ethylethers, substituted benzyl ethers and esters. In some embodiments, theprotecting group at R₅ can be an amine protecting group such as, e.g.,carbamates and substituted carbamates. In one embodiment, the protectinggroup at any of R₁, R₂, R₅, R₆, and R₇, is selected from levulinoyl,benzyl (Bn), benzoyl (Bz), methoxybenzyl (MPM), azide, allyl and silylether protecting group (e.g., tBDMS or tBDPS), as long as two, three,four or more of the protecting groups are orthogonal protecting groups.In one embodiment, R₁, R₂, R₅, R₆, and R₇ are protecting groups selectedfrom levulinoyl, benzyl, benzoyl, MPM, azide, allyl and silyl etherprotecting group (e.g., tBDMS or tBDPS), and none of R₁, R₂, R₅, R₆, orR₇ are the same protecting group.

In one embodiment, R₃ and R₄ are protecting groups, e.g., selected frombenzoyl and Fmoc, and, e.g., R₃ and R₄ are not the same protectinggroup.

In one embodiment, the protecting groups can be: R₁ is levulinoyl, R₂ isbenzyl, R₃ is benzoyl, R₄ is Fmoc, R₅ is azide, R₆ is allyl, and R₇ istBDPS; R₁ is levulinoyl, R₂ is benzyl, R₃ is benzoyl, R₄ is Fmoc, R₅ isNHCBz, R₆ is allyl, and R₇ is methoxybenzyl; R₁ is benzoyl, R₂ isbenzyl, R₃ is benzoyl, R₄ is Fmoc, R₅ is azide, R₆ is allyl, and R₇ istBDPS; R₁ is benzoyl, R₂ is benzyl, R₃ is benzoyl, R₄ is Fmoc, R₅ isNHCBz, R₆ is allyl, and R₇ is methoxybenzyl; R₁ is levulinoyl, R₂ isbenzyl, R₃ is benzoyl, R₄ is tBDMS, R₅ is azide, R₆ is benzyl and R₇ ismethoxybenzyl; or R₁ is benzoyl, R₂ is benzyl, R₃ is levulinoyl, R₄ istBDMS, R₅ is azide, R₆ is benzyl, and R₇ is methoxybenzyl.

In one embodiment, the disaccharide is a protected disaccharidedescribed herein, e.g., the disaccharide is a disaccharide provided inTable I, Table II, FIG. 6, FIG. 7, FIG. 14 or FIG. 15.

In one aspect, the disclosure features an oligosaccharide made partiallyor entirely of the diversely protected disaccharides described herein.Moieties protected by a class of protecting group, e.g., R₁, can bederivatized, e.g., with a sulfate moiety, or with a hydrogen orhydrogens. The oligosaccharide can be a trisaccharide, atetrasaccharide, a pentasaccharide, a hexasaccharide, an octasaccharide,a decasaccharide, a dodecasaccharide, tetradecasaccharide,hexadecasaccharide, or octadecasaccharide.

In one embodiment, the oligosaccharide comprises a disaccharide havingthe structure of:

wherein R₈ is an alkyl group, e.g., an ethyl, methyl, propyl, butyl,pentyl, etc. group, and wherein each of R₁, R₂, R₅, R₆, and R₇ aredistinct from one another or a subset of R₁, R₂, R₅, R₆, and R₇ aredistinct from one another, e.g., three or more of these positions aredistinct from each other. In one embodiment, R₁, R₂, R₅, R₆, and R₇, areorthogonal protecting groups and thus, each protecting group, R₁, R₂,R₅, R₆, and R₇, is selected such that any one can be individuallyremoved, without removing the others, to allow reaction of the protectedposition with another moiety, e.g., to result in the placement of asubstituent, e.g., a sulfate, acetate or a hydrogen, at the protectedmoiety. For example, the protecting group at R₂ can be removed withoutremoving R₁, R₅, R₆, and R₇.

In one embodiment, the protecting group at any of R₁, R₂, and R₆, and R₇can be a hydroxyl protecting group such as, e.g., silyl ethers, ethylethers, substituted benzyl ethers and esters. In some embodiments, theprotecting group at R₅ can be an amine protecting group such as, e.g.,carbamates and substituted carbamates. In one embodiment, the protectinggroup at any of R₁, R₂, R₅, R₆, and R₇, is selected from levulinoyl,benzyl (Bn), benzoyl (Bz), methoxybenzyl (MPM), azide, allyl and silylether protecting group (e.g., tBDMS or tBDPS), as long as two, three,four or more of the protecting groups are orthogonal protecting groups.In one embodiment, R₁, R₂, R₅, R₆, and R₇ are protecting groups selectedfrom levulinoyl, benzyl, benzoyl, MPM, azide, allyl and silyl etherprotecting group (e.g., tBDMS or tBDPS) and none of R₁, R₂, R₅, R₆, orR₇ are the same protecting group.

In one embodiment, the protecting groups can be: R₁ is levulinoyl, R₂ isbenzyl, R₅ is azide, R₆ is allyl, and R₇ is tBDPS; R₁ is levulinoyl, R₂is benzyl, R₅ is NHCBz, R₆ is allyl, and R₇ is methoxybenzyl; R₁ isbenzoyl, R₂ is benzyl, R₅ is azide, R₆ is allyl, and R₇ is tBDPS; or R₁is benzoyl, R₂ is benzyl, R₅ is NHCBz, R₆ is allyl, and R₇ ismethoxybenzyl.

In one aspect, the disclosure features a decasaccharide made partiallyor entirely of the diversely protected disaccharides described herein.These decasaccharides are of interest, in part, because they are of asize which can modulate biological activities.

In one embodiment, the decasaccharide comprises, e.g., consistsessentially of:

wherein each of X1, X2, X3 and X4 is independently A or B, and wherein

and wherein R₈ for each occurrence of A or B is an alkyl group, e.g., anethyl, methyl, propyl, butyl, pentyl, etc. group and wherein R₁, R₂, R₅,R₆, and R₇ for each occurrence of A or B each of R₁, R₂, R₅, R₆, and R₇are distinct from one another or a subset of R₁, R₂, R₅, R₆, and R₇ aredistinct from one another, e.g., three or more of these positions aredistinct from each other. In one embodiment, for each occurrence of A orB none of R₁, R₂, R₅, R₆, or R₇ within a single A or B is the same asanother protecting group within that same A or B. In embodiments theselection of one or more of R₁, R₂, R₅, R₆, and R₇ can differ between afirst and second group A, an A and B, or a first and second B.

In one embodiment, the protecting group at any of R₁, R₂, and R₆, and R₇can be a hydroxyl protecting group such as, e.g., silyl ethers, ethylethers, substituted benzyl ethers and esters. In some embodiments, theprotecting group at R₅ can be an amine protecting group such as, e.g.,carbamates and substituted carbamates. In one embodiment, the protectinggroup at any of R₁, R₂, R₅, R₆, and R₇, is selected from levulinoyl,benzyl (Bn), benzoyl (Bz), methoxybenzyl (MPM), azide, allyl and silylether protecting group (e.g., tBDMS or tBDPS), as long as two, three,four or more of the protecting groups are orthogonal protecting groups.In one embodiment, R₁, R₂, R₅, R₆, and R₇ are protecting groups selectedfrom levulinoyl, benzyl, benzoyl, MPM, azide, allyl and silyl etherprotecting group (e.g., tBDMS or tBDPS), and none of R₁, R₂, R₅, R₆, orR₇ are the same protecting group.

In one embodiment, the protecting groups can be: R₁ is levulinoyl, R₂ isbenzyl, R₅ is azide, R₆ is allyl, and R₇ is tBDPS; R₁ is levulinoyl, R₂is benzyl, R₅ is NHCBz, R₆ is allyl, and R₇ is methoxybenzyl; R₁ isbenzoyl, R₂ is benzyl, R₅ is azide, R₆ is allyl, and R₇ is tBDPS; or R₁is benzoyl, R₂ is benzyl, R₅ is NHCBz, R₆ is allyl, and R₇ ismethoxybenzyl.

In one embodiment, the decasaccharide comprises, e.g., consistingessentially of:

wherein each of X5, X6, X7 and X8 is independently C or D, and wherein

and wherein R₈ for each occurrence of C or D is an alkyl group, e.g., anethyl, methyl, propyl, butyl, pentyl, etc. group and wherein R₁, R₂, R₅,R₆, and R₇ for each occurrence of C or D each of R₁, R₂, R₅, R₆, and R₇are distinct from one another or a subset of R₁, R₂, R₅, R₆, and R₇ aredistinct from one another, e.g., three or more of these positions aredistinct from each other. In one embodiment, for each occurrence of C orD none of R₁, R₂, R₅, R₆, or R₇ within a single C or D is the same asanother protecting group within that same C or D. In embodiments theselection of one or more of R₁, R₂, R₅, R₆, and R₇ can differ between afirst and second group C, a C and D, or a first and second D.

In one embodiment, the protecting group at any of R₁, R₂, and R₆, and R₇can be a hydroxyl protecting group such as, e.g., silyl ethers, ethylethers, substituted benzyl ethers and esters. In some embodiments, theprotecting group at R₅ can be an amine protecting group such as, e.g.,carbamates and substituted carbamates. In one embodiment, the protectinggroup at any of R₁, R₂, R₅, R₆, and R₇, is selected from levulinoyl,benzyl (Bn), benzoyl (Bz), methoxybenzyl (MPM), azide, allyl and tBDPS,as long as two, three, four or more of the protecting groups areorthogonal protecting groups. In one embodiment, R₁, R₂, R₅, R₆, and R₇are protecting groups selected from levulinoyl, benzyl, benzoyl, MPM,azide, allyl and tBDPS and none of R₁, R₂, R₅, R₆, or R₇ are the sameprotecting group.

In one embodiment, the protecting groups can be: R₁ is levulinoyl, R₂ isbenzyl, R₅ is azide, R₆ is allyl, and R₇ is tBDPS; R₁ is levulinoyl, R₂is benzyl, R₅ is NHCBz, R₆ is allyl, and R₇ is methoxybenzyl; R₁ isbenzoyl, R₂ is benzyl, R₅ is azide, R₆ is allyl, and R₇ is tBDPS; or R₁is benzoyl, R₂ is benzyl, R₅ is NHCBz, R₆ is allyl, and R₇ ismethoxybenzyl.

In one aspect, the disclosure features a tetrasaccharide, e.g., atetrasaccharide shown in FIG. 16.

In one aspect, the disclosure features a hexasaccharide, e.g., ahexasaccharide shown in FIG. 17.

In one aspect, the disclosure features an oligosaccharide, e.g., adecasaccharide, that includes the following structure:

In one aspect, the disclosure features a decasaccharide having thefollowing structure:

In another aspect, the disclosure features a composition comprising aplurality of decasaccharides, wherein at least 20%, 30%, 40%, 50%, 60%,70%, 80%, 85%, 90%, 95%, 99% or 100% of the decasaccharides have thefollowing structure:

In one embodiment, all of the decasaccharides in the composition havethe following structure:

In one aspect, the disclosure features a diversely protectedmonosaccharide. The monosaccharide can be used to make larger saccharidestructures.

In one embodiment, the monosaccharide has the following structure:

wherein R₈ is a hydrogen or an alkyl group, e.g., an ethyl, a methyl, apropyl, a butyl, a pentyl, etc. and wherein R₁, R₂ group are orthogonalprotecting groups. In one embodiment, R₃ is a protecting group, e.g., aprotecting group selected from benzoyl or Fmoc.

In one embodiment, the protecting group at any of R₁ and R₂ can be ahydroxyl protecting group such as, e.g., silyl ethers, ethyl ethers,substituted benzyl ethers and esters, so long as the protecting groupsat R₁ and R₂ are orthogonal protecting groups. In one embodiment, theprotecting group at any of R₁ and R₂ is selected from levulinoyl, benzyl(Bn), benzoyl (Bz), methoxybenzyl (MPM), allyl and tBDPS, so long as theprotecting groups at R₁ and R₂ are orthogonal protecting groups.

In one embodiment, the protecting groups at positions R₁, R₂, and,optionally R₃ are different from protecting groups of a glucosamine thathas a protecting group at one or more of: attached to an oxygen atposition C1 of the glucosamine; attached to position C2 of theglucosamine; attached to an oxygen at position C3 of the glucosamine;and attached to an oxygen at position C6 of the glucosamine.

In one embodiment, the monosaccharide has one of the followingstructures:

In one embodiment, the monosaccharide has the following structure:

wherein each of R₅, R₆, and R₇ are distinct from one another or a subsetof R₅, R₆, and R₇ are distinct from one another, e.g., two or more ofthese positions are distinct from each other. In one embodiment, R₅, R₆,and R₇, are orthogonal protecting groups and thus, each protectinggroup, R₅, R₆, and R₇, is selected such that any one can be individuallyremoved, without removing the others, to allow reaction of the protectedposition with another moiety, e.g., to result in the placement of asubstituent, e.g., a sulfate or acetate, at the protected moiety. Forexample, the protecting group at R₅ can be removed without removing R₆,and R₇.

In one embodiment, the protecting group at any of R₆ and R₇ can be ahydroxyl protecting group such as, e.g., silyl ethers, ethyl ethers,substituted benzyl ethers and esters. In some embodiments, theprotecting group at R₅ can be an amine protecting group such as, e.g.,carbamates and substituted carbamates. In one embodiment, the protectinggroup at any of R₅, R₆, and R₇, is selected from levulinoyl, benzyl(Bn), benzoyl (Bz), methoxybenzyl (MPM), azide, allyl and silyl etherprotecting group (e.g., tBDMS or tBDPS), as long as two or more of theprotecting groups are orthogonal protecting groups. In one embodiment,R₅, R₆, and R₇ are protecting groups selected from levulinoyl, benzyl,benzoyl, MPM, azide, allyl and silyl ether protecting group (e.g., tBDMSor tBDPS), and none of R₅, R₆, or R₇ are the same protecting group.

In one embodiment, R₄ is a protecting group, e.g., selected from benzoyland Fmoc.

In one embodiment, the protecting groups at position R₅, R₆, R₇ andoptionally R₄ are different from protecting groups of an uronic acidthat has a protecting group at one or more of: attached to an oxygen atposition C2 of the uronic acid; and attached to an oxygen at position C3of the uronic acid.

In one embodiment, the monosaccharide has one of the followingstructures:

In one embodiment, the monosaccharide is a monosaccharide provided inany of FIGS. 1-5, 12 and 13.

In another aspect, the disclosure features a method of making anoligosaccharide that is a disaccharide or larger, e.g., a sequence ofsaccharide structures having a preselected pattern of derivatization.Embodiments of the method allow the design and synthesis ofoligosaccharide structures having preselected complex patterns ofderivatization, e.g., preselected complex patterns of sulfation oracetylation. Saccharide structures or subunits, each having theappropriate pattern of protecting groups, are joined together to allowthe production of the larger saccharide structure having the preselectedpattern of derivatization. Each saccharide structure can be a diverselyprotected saccharide structure, e.g., a diversely protected saccharidestructure described herein. The library provides a plurality ofoligosaccharide structures having diverse patterns of the protectinggroups. Thus, one can select a first library member having a pattern ofprotecting groups which, upon deprotection of a particular protectinggroup can give a selected pattern of substituents, e.g., sulfate,acetate and hydrogen. As referred to above, the first library member isjoined to one or more subsequent library members having selectedpatterns of protecting groups and selected to provide a pattern ofprotecting groups. As referred to above, deprotection reactions can beused to removed a particular protecting group within the oligosaccharideand add a substituent at the deprotected position, while maintaining theorthogonal protecting groups at other positions within theoligosaccharide to provide a preselected pattern of substituents, e.g.,sulfate, acetate or hydrogen. The oligosaccharide can be, e.g., adisaccharide, a trisaccharide, a tetrasaccharide, a pentasaccharide, ahexasaccharide, an octasaccharide, a decasaccharide, a dodecasaccharide,tetradecasaccharide, hexadecasaccharide, or octadecasaccharide.

The method includes:

providing a first diversely protected saccharide structure, wherein saidsaccharide structure is a monosaccharide or larger;

providing a second saccharide structure, wherein said second saccharidestructure is a monosaccharide or larger, and optionally is a diverselyprotected saccharide structure; and

attaching said first and second saccharide structures,

thereby making an oligosaccharide of preselected sequence.

In one embodiment, the method further includes providing a thirdsaccharide structure, wherein the third saccharide structure is amonosaccharide or larger; and attaching the third saccharide structureto the saccharide structure formed from the first and second saccharidestructure. In one embodiment, the third saccharide structure is adiversely protected saccharide structure. In one embodiment, the methodcan further include providing and attaching a fourth, fifth, sixth,seventh, etc. saccharide structure to make the oligosaccharide. Any ofthe fourth, fifth, sixth, seventh, etc. saccharide structures can be,e.g., a diversely protected saccharide structure.

In one embodiment, the saccharide structure (e.g., the first, second,third, fourth, etc. saccharide structure) is a monosaccharide, adisaccharide, a tetrasaccharide, a pentasaccharide, a hexasaccharide, anoctasaccharide, or a decasaccharide. In one embodiment, the firstsaccharide structure is a disaccharide and the second saccharidestructure is selected from a monosaccharide, a disaccharide, atrisaccharide, a tetrasaccharide, a pentasaccharide, a hexasaccharide, aheptasaccharide, and an octasaccharide.

In one embodiment, the saccharide structure (e.g., the first, second,third, fourth, etc. saccharide structure) is a monosaccharide describedherein or a disaccharide a disaccharide described herein.

In one embodiment, the method includes making a disaccharide comprising

providing a first diversely protected monosaccharide described hereinand a second diversely protected monosaccharide described herein; andattaching the first monosaccharide to the second monosaccharide, tothereby make the disaccharide.

In one embodiment, the method includes providing a first disaccharidestructure, e.g., a diversely protected disaccharide described herein;attaching a second disaccharide structure, e.g., a diversely protecteddisaccharide structure described herein, to the first disaccharidestructure to provide a first tetrasaccharide structure; providing athird disaccharide structure, e.g., a diversely protected disaccharidedescribed herein; attaching a fourth disaccharide structure, e.g., adiversely protected disaccharide structure described herein, to thethird disaccharide structure to provide a second tetrasaccharidestructure; attaching the first tetrasaccharide structure to the secondtetrasaccharide structure to provide an octasaccharide structure; andattaching a fifth disaccharide structure, e.g., a diversely protecteddisaccharide structure described herein, to the octasaccharide structureto thereby provide a decasaccharide structure.

In one embodiment, the method further includes deprotecting one or morepositions within the saccharide structure or saccharide structures thatattaches one saccharide structure to another to form an unprotectedmoiety or moieties. The deprotected moiety or moieties can then be usedto attach one saccharide structure to another. In one embodiment, onesaccharide structure, e.g., the first saccharide structure, is attachedto another saccharide structure, e.g., the second saccharide structure,using a reaction mixture that comprises a catalyst, e.g., TMSOTf orTESOTf.

In one embodiment, the oligosaccharide is an oligosaccharide describedherein.

In an embodiment, the method is repeated to form a collection or libraryof oligosaccharides.

In one embodiment, the method further comprises deprotecting aprotecting group while maintaining orthogonal protecting groups at otherpositions to form an unprotected moiety or moieties. The method canfurther comprise forming substituents, e.g., sulfate, acetate, at thedeprotected position or positions.

In an embodiment, the method is repeated to form a collection or libraryof oligosaccharides having preselected sequences.

In one aspect, the disclosure features a method of making anoligosaccharide structure having a defined level or pattern ofderivatization, e.g., sulfation, comprising:

providing a diversely protected oligosaccharide structure, e.g., adiversely protected oligosaccharide structure described herein, andoptionally of preselected sequence;

deprotecting a class of protected positions in the oligosaccharide,e.g., an oligosaccharide described herein, to form unprotected moietiesand forming substituent moieties, e.g., sulfate moieties or acetatemoieties, on the deprotected positions.

In one embodiment the method further includes:

deprotecting a subsequent second class of protected positions in theoligosaccharide, e.g., an oligosaccharide described herein, to form asecond class of unprotected moieties and forming substituent moieties,e.g., sulfate moieties, acetate moieties or hydrogen moieties, on thesecond class of deprotected positions.

In one embodiment the method further includes:

deprotecting a subsequent (e.g., a third or fourth) class of protectedpositions in the oligosaccharide, e.g., an oligosaccharide describedherein, to form a subsequent class of unprotected moieties, and formingsubstituent moieties, e.g., sulfate moieties, acetate moieties, on thesubsequent class of deprotected positions.

In an embodiment the method is repeated to form a library ofoligosaccharides having preselected levels of patterns of substituents,e.g., sulfation, acetylation.

Although deprotection is sometimes referred to separately, it isunderstood that deprotection and derivatization can occur sequentiallyor simultaneously.

In one aspect, the disclosure features method of making a diverselyprotected monosaccharide, e.g., a diversely protected monosaccharidedescribed herein, comprising:

providing a glucose;

attaching a first protecting group selected from levulinoyl, allyl,benzyl, benzoyl, azide, NHCBz, tBDPS, tBDMS or methoxybenzyl to anoxygen attached to position C3 of the glucose forming a uronic acid fromthe glucose;

attaching a second protecting group selected from levulinoyl, allyl,benzyl, azide, NHCBz, tBDPS, tBDMS or methoxybenzyl to an oxygenattached to position C2 of the uronic acid, wherein the first protectinggroup differs from the second protecting group.

In one embodiment, the method includes:

providing a glucose;

attaching a benzyl group at an oxygen attached to position C3 of theglucose;

forming a uronic acid from the glucose; and

attaching a levulinoyl or benzoyl to an oxygen attached to position C2of the uronic acid, to thereby make the monosaccharide.

In one embodiment, the method includes one or more of the stepsdescribed in FIG. 1, 3, 5 or 13.

In one aspect, the disclosure features a method of making amonosaccharide, e.g., a diversely protected monosaccharide describedherein, comprising:

providing a glucosamine;

attaching a first protecting group selected from levulinoyl, allyl,benzyl, azide, benzoyl, NHCBz, tBDPS, tBDMS or methoxybenzyl at positionC2 of the glucosamine;

attaching a second protecting group selected from levulinoyl, allyl,benzyl, azide, benzoyl, NHCBz, tBDPS, tBDMS or methoxybenzyl to anoxygen at position C3 of the glucosamine; and

attaching a third protecting group selected from levulinoyl, allyl,benzyl, azide, benzoyl, NHCBz, tBDPS, tBDMS or methoxybenzyl to anoxygen at position C6 of the glucosamine, wherein the first, second andthe third protecting groups all differ from each other to thereby makethe monosaccharide.

In one embodiment, the method includes:

providing a glucosamine;

attaching an N₃ at position C2 of the glucosamine;

attaching an allyl to an oxygen at position C3 of the glucosamine; and

attaching a tBDPS, tBDMS or MPM to an oxygen at position C6 of theglucosamine, to

thereby make the monosaccharide.

In one embodiment, the method includes one or more of the stepsdescribed in FIG. 2, 4 or 12.

In one aspect, the disclosure features a composition that includes asaccharide structure described herein, e.g., a monosaccharide,disaccharide or larger oligosaccharide described herein. In oneembodiment, the composition can further include a diluent, excipient orcarrier. In one embodiment, the composition is dried or lyophilized.

In one aspect, the disclosure features a compound described any of FIG.1, 2, 3, 4, 5, or 12-18.

In one aspect, the disclosure features a collection or library ofsaccharide structures, e.g., saccharide structures described herein,e.g., a monosaccharide, disaccharide or larger oligosaccharide describedherein.

In one aspect, the disclosure features preparations, e.g., substantiallypurified preparations, e.g., pharmaceutical preparations, of one or moresaccharide structure described herein, e.g., a monosaccharide,disaccharide or larger oligosaccharide described herein. Also within thedisclosure are reaction mixtures having two or more of the saccharidestructures described herein, e.g., a monosaccharide, disaccharide orlarger oligosaccharide described herein.

In one aspect, disclosure features oligosaccharides (either singly or ascollections or libraries) are provided as a plurality of substantiallypurified preparations. In an embodiment each partially purifiedpreparation is free of substantial amounts of other protectedoligosaccharides or of substantial amounts of other protectedoligosaccharides of the same length. Also within the disclosure arereaction mixtures having two or more of the oligosaccharides describedherein.

In one aspect, the disclosure features a method of analyzing anoligosaccharide, e.g., an oligosaccharide described herein, comprising:

providing a test oligosaccharide, e.g., an oligosaccharide describedherein having a predetermined level or pattern of derivatization;

determining a property of said test oligosaccharide,

thereby analyzing an oligosaccharide.

In one aspect, the disclosure features a method of identifying anoligosaccharide, e.g., an oligosaccharide described herein, that bindsto a target protein, comprising:

providing a test oligosaccharide, a collection or a library of testoligosaccharides having a predetermined level of pattern ofderivatization;

determining if the oligosaccharide or one or more of the oligosaccharidefrom the collection or library binds to a target polypeptide, to therebyidentify an oligosaccharide that binds to the target polypeptide.

In one aspect, the disclosure features a database disposed on tangiblemedium that includes: at least 10 records wherein a record comprises, anidentifier which identifies a saccharide structure disclosed herein; andoptionally an identifier which identifies a biological or chemicalproperty of the saccharide structure.

Aspects of the disclosure also include a system comprising: a userinterface for inputting a query; a processor for generating a queryresult; a selector to select a parameter based on the sequence, achemical or a biological property of a saccharide structure disclosedherein; and the database described above.

The invention allows for the production of oligosaccharide structures,e.g., a decasaccharide, having defined structure and/or properties. Theinvention provides for oligosaccharide drugs and drug candidates, e.g.,drugs or drug candidates having a desired biological property, e.g.,anti-factor IIa activity, anti-factor Xa activity, anti-thromboticactivity, anti-inflammatory activity, anti-angiogenic activity,anti-cancer or anti-metastatic activity. Preparations of theoligosaccharides, e.g., preparations of oligosaccharide drugs, can haveoptimized heterogeneity and can, e.g., be less heterogeneous thanoligosaccharide drugs prepared from natural sources. The invention alsoprovides libraries and other constructs useful for the production ofoligosaccharides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts synthesis of an iduronic acid having a first protectinggroup that allows derivatization or does not allow for derivatization(10) and an iduronic acid that has a first protecting group that allowsfor derivatization and a second protecting group that does not allow forderivatization (8).

FIG. 2 depicts synthesis of a glucosamine having a first protectinggroup that allows derivatization or does not allow for derivatization(17, 18, 23 and 24) and a glucosamine that has a first protecting groupthat allows for derivatization and a second protecting group that doesnot allow for derivatization (15, 16, 21 and 22).

FIG. 3 depicts synthesis of a diversely protected iduronic acid havingorthogonal protecting groups of a Lev at C-2 in Compound 26 and a Bz atC-2 in Compound 28.

FIG. 4 depicts synthesis of a diversely protected glucosamine havingorthogonal protecting groups of a tBDPS in Compound 36 and MPM inCompound 34; and an azide in Compound 36 and a benzyl carbamate NHCBz inCompound 34.

FIG. 5 depicts synthesis of a diversely protected glucuronic acid havingorthogonal protecting groups of a Lev at C-2 in Compound 40 and a Bz atC-2 in Compound 42.

FIG. 6 depicts the synthesis of various diversely protecteddisaccharides by various combinations of diversely protectedmonosaccharides of iduronic acid and glucosamine.

FIG. 7 depicts the synthesis of various diversely protecteddisaccharides by various combinations of diversely protectedmonosaccharides of glucuronic acid and glucosamine.

FIG. 8 shows a diversely protected decasaccharide having a preselectedsequence and different conditions for deprotecting and addingsubstituents at C-2 of iduronic acid within the diversely protecteddecasaccharide.

FIG. 9 shows a diversely protected decasaccharide having a preselectedsequence and different conditions for deprotecting and addingsubstituents at C-3 of Glucosamine/and Iduronic acid residues within thediversely protected decasaccharide.

FIG. 10 shows a diversely protected decasaccharide having a preselectedsequence and different conditions for deprotecting and addingsubstituents at C-6 of glucosamine residues within the diverselyprotected decasaccharide.

FIG. 11 shows a diversely protected decasaccharide having a preselectedsequence and different conditions for deprotecting and addingsubstituents at C-2 of glucosamine residues within the diverselyprotected decasaccharide.

FIG. 12 depicts the synthesis of a diversely protected Glucosaminesynthon.

FIG. 13 depicts the synthesis of diversely protected Iduronic acidsynthons.

FIG. 14 depicts the synthesis of a diversely protected disaccharide.

FIG. 15 depicts the synthesis of a diversely protected disaccharide.

FIG. 16 depicts the synthesis of a diversely protected tetrasaccharide.

FIG. 17 depicts the synthesis of a diversely protected hexasaccharide.

FIG. 18 depicts the synthesis of a monosulfated disaccharide.

DETAILED DESCRIPTION

Described in the present disclosure are oligosaccharides that are adisaccharide or larger that provide a scaffold for makingoligosaccharides having a sequence with a preselected pattern ofsubstituents, e.g., sulfates, acetates, and hydrogen, at positionswithin the oligosaccharide that are amendable to derivatization. Theseoligosaccharides allow the design and synthesis of oligosaccharidestructures having preselected complex patterns of derivatization, e.g.,preselected complex patterns of sulfation or acetylation. In someembodiments, the oligosaccharide has only two or three differentprotecting groups. At least two of the protecting groups have differentreactivities. One protecting group is replaced to a first degree, e.g.,substantially completely replaced, with a derivatizing group underselected conditions. The other protecting group is replaced to a seconddegree, usually relatively less, e.g., it gives substantially noderivatization, under the same conditions.

In other embodiments, the positions amenable to derivatization within adisaccharide or a disaccharide of a larger oligosaccharide are alldistinct from one another or a subset of the positions amendable toderivatization within the disaccharide are distinct from one another,e.g., three or more of these positions are distinct from each other. Inone embodiment, the positions amendable to derivatization within adisaccharide are orthogonal protecting groups and thus, each protectinggroup is selected such that any one can be individually removed, withoutremoving the others, to allow reaction of the protected position withanother moiety, e.g., to result in the placement of a substituent, e.g.,a sulfate, acetate or hydrogen, at the protected moiety. Theseoligosaccharides allow for placement of a particular substituent atparticular positions with an oligosaccharide.

Also described in the present disclosure are monosaccharides that can beused, e.g., to make oligosaccharide structures having protecting groupsat positions amenable to derivatization, methods of making a saccharidestructure described herein, e.g., a monosaccharide, disaccharide orlarger oligosaccharide described herein. Collections and libraries of asaccharide structure, e.g., a monosaccharide, disaccharide or largeroligosaccharide described herein, kits, reaction mixtures, andcompositions are described herein. Furthermore, methods of analyzing anoligosaccharide described herein, are provided.

Methods, compounds and compositions described herein can use, or be madewith, two protecting groups. The two protecting groups can, and usuallywill, have substantially different reactivities (the ability to bereplaced with a derivative) under a given set of conditions. In mostcases one protecting group will be replaced to a substantially greaterdegree than the other under a selected condition. In embodiments theother protecting group will be substantially more reactive under asecond set of conditions. In some embodiments one protecting group willbe substantially completely replaced under a selected condition and theother protecting group will be substantially unreacted (not replaced)under those conditions. In embodiments both groups will be reactive,either to the same degree, or more commonly, to different degrees, undera selected condition. The latter relationship is useful, e.g., in makinglibraries, e.g., combinatorial libraries, or in dirty synthesis.

The term “substituent” as used herein refers to any moiety naturallyassociated with an oligosaccharide at a position amendable toderivatization. For example, the substituent is a sulfate, an acetate ora hydrogen.

Positions amendable to derivatization include any position on asaccharide structure that can have a sulfate or acetate associated withthat position. Positions involved with linking one saccharide structureto another are not encompassed by this term.

The term “derivatization” includes sulfation and acetylation but doesnot include hydrogenation and linkage of saccharide structures to eachother.

A “collection” as used herein means more than one and less than tenmembers. For example, a collection can be 2, 3, 4, 5, 6, 7, 8 or 9monosaccharides or oligosaccharides.

The term “library” as used herein refers to 10 or more members. Forexample, a library can include at least 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 500, or 1,000monosaccharides or oligosaccharides.

Monosaccharide Synthesis

Described herein are monosaccharides having a protecting group at eachposition in the monosaccharide amenable to derivatization. These areuseful, e.g., for providing disaccharides or larger oligosaccharides, orlibraries thereof, having preselected sequences and/or levels orpatterns of derivatization, e.g., sulfation or acetylation.

In some aspects, the protecting group at any given position can be afirst protecting group that allows derivatization, e.g., sulfation oracetylation, or a second protecting group that does not allowderivatization, e.g., sulfation or acetylation. The identity of eachprotecting group at positions amendable to derivatization canindependent of the protecting group at any other position amendable toderivatization.

Examples of such monosaccharides are provided in FIGS. 1 and 2. Inaddition, examples of methods of synthesizing these monosaccharides areprovided in FIGS. 1 and 2. Known methods can be used to make othermonosaccharides, e.g., monosaccharides described herein.

In other embodiments, diversely protected monosaccharides are described.A diversely protected monosaccharide refers to a monosaccharide havingeach protecting group or more than two protecting groups of themonosaccharide that are orthogonal protecting groups and thus, eachprotecting group, or a subset of the protecting groups (i.e., more thantwo protecting groups within a monosaccharide) is selected such that anyone can be individually removed, without removing the others, to allowreaction of the protected position with another moiety, e.g., to resultin the placement of a substituent, e.g., a sulfate, acetate or ahydrogen, at the protected moiety.

Examples of diversely protected monosaccharides are provided in FIGS.3-5, 12 and 13. FIGS. 3-5, 12 and 13 also describe exemplary methods ofsynthesizing these diversely protected monosaccharides. Known methodscan be used to make other monosaccharides, e.g., other monosaccharidesdescribed herein

Disaccharide Synthesis

The disclosure also provides disaccharides having a protecting group ateach position in the disaccharide amenable to derivatization. Theprotecting group at any given position can be a first protecting groupthat allows derivatization, e.g., sulfation or acetylation, or a secondprotecting group that does not allow derivatization, e.g., sulfation oracetylation. These disaccharides can be made by combiningmonosaccharides having protecting groups at positions amendable toderivatization with each other to form a disaccharide. In one aspect,various combinations of protected monosaccharides, e.g., the protectedmonosaccharides described herein, can be made to form a collection orlibrary of protected disaccharides.

A disaccharide, e.g., a disaccharide described herein, can be made usinga standard TMSOTf-mediated coupling of one protected monosaccharide toanother protected monosaccharide. For example, an uronic acidtrichloroacetimidate donor and a glucosamine acceptor can be coupled toone another using known TMSOTf-coupling techniques. Such techniques aredescribed in Lohman et. al. (2004) J. Org. Chem. 69(12), 4081-4093.

In some embodiments, the disaccharide can have only two or threedifferent protecting groups. The two or at least two of the threeprotecting groups have different reactivities. One protecting group isreplaced to a first degree, e.g., substantially completely replaced orcompletely, with a derivatizing group under selected conditions. Thisprotecting group is referred to herein as “a protecting group thatallows derivatization”. The other protecting group gives relativelyless, e.g., it gives substantially no derivatization or noderivatization, under the same conditions. This protecting group isreferred to herein as “a protecting group that does not allowderivatization”. It should be noted that if the reactive conditions arechanged, a protecting group that allows derivatization under one set ofconditions can be a protecting group that does not allow derivatizationunder another set of conditions.

The protecting groups can be selected from known protecting groups suchas the protecting groups described herein.

Table I and schematic A provided below exemplify various disaccharidesthat can be produced by coupling monosaccharides protected with variouscombinations of a protecting group that allows derivatization (e.g.,benzoyl and/or a benzoyl containing group) and monosaccharides protectedby a protecting group that does not allow derivatization (e.g., benzyland/or azide). Monosaccharides are referred to in Table I as AA, AB andBB. The disaccharides are an uronic acid (iduronic acid) coupled to ahexosamine (glucosamine). However, it should be understood that othercombinations, for example, a glucuronic acid and a glucosamine, can beproduced using the same methodology. The disaccharides exemplified arevarious combinations of the monosaccharides AA, AB and BB. For uronicacid, A is a benzoyl protecting group that allows derivatization, e.g.,when derivatized, the benzoyl will be replaced with SO₃—, and B is abenzyl protecting group that does not allow derivatization, e.g., thebenzyl will be replaced with a hydrogen. For glucosamine, at positionsR₆ and R₇, A represents a benzoyl protecting group that allowsderivatization, e.g., when derivatized, the benzoyl will be replacedwith SO₃—, and B represents a benzyl protecting group that does notallow derivatization, e.g., the benzyl will be replaced with a hydrogen.For position R₅ of glucosamine, A represents a benzoyl containing group,NHCBz, as the protecting group that allows derivatization, e.g., whenderivatized, the benzoyl containing group will be replaced with NHSO₃—,and B represents an azide protecting group that does not allowderivatization, e.g., the azide will be replaced with NH₂.

Table I provides various combinations of disaccharides that haveprotecting groups at positions within the monosaccharides (theglucuronic acid and glucosamine) amendable to derivatization. Thedisaccharide code denotes the relevant substituents in the orderR¹—R⁷—R⁶—R⁵.

TABLE I Exemplary Uronic Acid-Hexosamine Disaccharides Disacc. Code R1R2 R3 R4 R5 R6 R7 U + H 1 AAAB Bz Bn Lev Bz N3 Bz Bz  9 + 17 2 AAAA BzBn Lev Bz NHCBz Bz Bz  9 + 18 3 BAAB Bn Bn Lev Bz N3 Bz Bz 11 + 17 4BAAA Bn Bn Lev Bz NHCBz Bz Bz 11 + 18 5 ABAB Bz Bn Lev Bz N3 Bz Bn  9 +15 6 ABAA Bz Bn Lev Bz NHCBz Bz Bn  9 + 16 7 AABB Bz Bn Lev Bz N3 Bn Bz 9 + 21 8 AABA Bz Bn Lev Bz NHCBz Bn Bz  9 + 22 9 BBAB Bn Bn Lev Bz N3Bz Bn 11 + 15 10 BBAA Bn Bn Lev Bz NHCBz Bz Bn 11 + 16 11 ABBB Bz Bn LevBz N3 Bn Bn  9 + 23 12 ABBA Bz Bn Lev Bz NHCBz Bn Bn  9 + 24 13 BBBB BnBn Lev Bz N3 Bn Bn 11 + 23 14 BBBA Bn Bn Lev Bz NHCBz Bn Bn 11 + 24 15BABB Bn Bn Lev Bz N3 Bn Bz 11 + 21 16 BABA Bn Bn Lev Bz NHCBz Bn Bz 11 +22

Table II and schematic B provided below exemplify additionaldisaccharides that can be produced by coupling monosaccharides protectedwith various combinations of a protecting group that allowsderivatization (e.g., benzoyl or a benzoyl containing group) andmonosaccharides protected by a protecting group that does not allowderivatization (e.g., benzyl or azide). Monosaccharides are referred toin Table II as AA, AB and BB. The disaccharides are a hexosamine(glucosamine) coupled to an uronic acid (iduronic acid). However, itshould be understood that other combinations, for example, a glucosaminecoupled to a glucuronic acid, can be produced using the samemethodology. The disaccharides exemplified are various combinations ofthe monosaccharides AA, AB and BB. For glucosamine, at positions R₆ andR₇, A represents a benzoyl protecting group that allows derivatization,e.g., when derivatized, the benzoyl will be replaced with SO₃—, and Brepresents a benzyl protecting group that does not allow derivatization,e.g., the benzyl will be replaced with a hydrogen. For position R₅ ofglucosamine, A represents a benzoyl containing group, NHCBz, as theprotecting group that allows derivatization, e.g., when derivatized, thebenzoyl containing group will be replaced with NHSO₃—, and B representsan azide protecting group that does not allow derivatization, e.g., theazide will be replaced with NH₂. For uronic acid, A is a benzoylprotecting group that allows derivatization, e.g., when derivatized, thebenzoyl will be replaced with SO₃—, and B is a benzyl protecting groupthat does not allow derivatization, e.g., the benzyl will be replacedwith a hydrogen.

TABLE II Exemplary Hexosamine-Uronic Acid Disaccharides Disacc. Code R1R2 R3 R4 R5 R6 R7 H + U 1 AAAB Bz Bn tBDMS 2-Nap N₃ Bz Bz 17 + 8  2 AAAABz Bn tBDMS 2-Nap NHCBz Bz Bz 18 + 8  3 BAAB Bn Bn tBDMS 2-Nap N₃ Bz Bz17 + 10 4 BAAA Bn Bn tBDMS 2-Nap NHCBz Bz Bz 18 + 10 5 ABAB Bz Bn tBDMS2-Nap N₃ Bz Bn 15 + 8  6 ABAA Bz Bn tBDMS 2-Nap NHCBz Bz Bn 16 + 8  7AABB Bz Bn tBDMS 2-Nap N₃ Bn Bz 21 + 8  8 AABA Bz Bn tBDMS 2-Nap NHCBzBn Bz 22 + 8  9 BBAB Bn Bn tBDMS 2-Nap N₃ Bz Bn 15 + 10 10 BBAA Bn BntBDMS 2-Nap NHCBz Bz Bn 16 + 10 11 ABBB Bz Bn tBDMS 2-Nap N₃ Bn Bn 23 +8  12 ABBA Bz Bn tBDMS 2-Nap NHCBz Bn Bn 24 + 8  13 BBBB Bn Bn tBDMS2-Nap N₃ Bn Bn 23 + 10 14 BBBA Bn Bn tBDMS 2-Nap NHCBz Bn Bn 24 + 10 15BABB Bn Bn tBDMS 2-Nap N₃ Bn Bz 21 + 10 16 BABA Bn Bn tBDMS 2-Nap NHCBzBn Bz 22 + 10

As described elsewhere herein, these disaccharides are useful, forproviding oligosaccharides, or libraries thereof, having preselectedsequences and/or levels or patterns of derivatization, e.g., sulfationor acetylation.

oligosaccharide synthesis

The disclosure features oligosaccharides that can have a preselectedsequence, e.g., a sequence of saccharide structures having a preselectedpattern of derivatization. The oligosaccharide, e.g., an oligosaccharidedescribed herein, allows the design and synthesis of oligosaccharidestructures having preselected complex patterns of derivatization, e.g.,preselected complex patterns of sulfation or acetylation. Theoligosaccharide can be, e.g., a disaccharide, a trisaccharide, atetrasaccharide, a pentasaccharide, a hexasaccharide, an octasaccharide,a decasaccharide, a dodecasaccharide, tetradecasaccharide,hexadecasaccharide, or octadecasaccharide.

In some embodiments, the oligosaccharide includes one or moredisaccharide disclosed herein. Preferably, all of the disaccharide unitsof the oligosaccharide are disaccharide units which have a protectinggroup at all positions amendable to derivatization, e.g., all of thedisaccharides of the oligosaccharide are a disaccharide describedherein.

In some embodiments, the oligosaccharide can include a disaccharide ordisaccharides having only two or three different protecting groups. Theprotecting group at any given position within the disaccharide ordisaccharide can be a first protecting group that allows derivatization,e.g., sulfation or acetylation, or a second protecting group that doesnot allow derivatization, e.g., sulfation or acetylation. When theoligosaccharide includes more than one disaccharide unit describedherein, the identity of a protecting group at any position within thedisaccharide is independent of the identity of a protecting group in anyother disaccharide of the oligosaccharide. The disaccharide can be,e.g., a disaccharide depicted in Table I, Table II, FIG. 6, FIG. 7, FIG.14 or FIG. 15.

The protecting groups can be selected from known protecting groups suchas the protecting groups described herein.

The oligosaccharide can be made by providing a first saccharidestructure that is a monosaccharide or larger, e.g., the saccharidestructure described herein, e.g., a disaccharide described herein;providing a second saccharide structure, e.g., the saccharide structuredescribed herein, e.g., a disaccharide described herein, and attachingthe first saccharide structure to the second saccharide structure. Themethod can also include attaching a third, fourth, fifth, sixth,seventh, etc. saccharide structure to make an oligosaccharide, e.g., anoligosaccharide having a preselected sequence. In one embodiment, themethod includes providing a first disaccharide structure, e.g., adisaccharide described herein; attaching a second disaccharidestructure, e.g., a disaccharide structure described herein, to the firstdisaccharide structure to provide a first tetrasaccharide structure;providing a third disaccharide structure, e.g., a disaccharide describedherein; attaching a fourth disaccharide structure, e.g., a disaccharidestructure described herein, to the third disaccharide structure toprovide a second tetrasaccharide structure; attaching the firsttetrasaccharide structure to the second tetrasaccharide structure toprovide an octasaccharide structure; and attaching a fifth disaccharidestructure, e.g., a disaccharide structure described herein, to theoctasaccharide structure to thereby provide a decasaccharide structure.This is one example of how an oligosaccharide can be made. However,saccharide structures can be assembled by alternative methods. Forexample, a first disaccharide structure can be attached to a seconddisaccharide structure to form a tetrasaccharide. A third disaccharidecan be attached to the tetrasaccharide to provide a hexasaccharide. Afourth disaccharide can be attached to the hexasaccharide to form anoctasaccharide and a fifth disaccharide can be attached to theoctasaccharide to form a decasaccharide.

The methods for producing an oligosaccharide include the methodsdepicted in FIG. 16 and/or FIG. 17.

The saccharide structures can be attached to one another using astandard TMSOTf-mediated coupling of one protected monosaccharide toanother protected monosaccharide. For example, an uronic acidtrichloroacetimidate donor and a glucosamine acceptor can be coupled toone another using known TMSOTf-coupling techniques. Such techniques aredescribed in Lohman et. al. (2004) J. Org. Chem. 69(12), 4081-4093.

The saccharide structures used to assemble an oligosaccharide, e.g., anoligosaccharide described herein, can include one or more protectinggroups at a position within the saccharide structure that forms linkageswith another saccharide structure. This position can be deprotected andthe saccharide structures can be linked through this position of thesaccharide structure using, e.g., TMSOTf or TESOTf-coupling.

The following is an example of a method of assembling various saccharidestructures to prepare a protected oligosaccharide having a sequence thatallows for a preselected pattern of derivatization. In this example,disaccharides 1, 2, 3 and 4 from Table I above are used. However, it isunderstood that any combination of disaccharides, e.g., any combinationof the disaccharides depicted in Table I or Table II, can be used tomake an oligosaccharide. Disaccharides 1, 2, 3 and 4 of Table I have thefollowing structures:

1. (also referred to in Table I as AAAB)

2. (also referred to in Table I as AAAA)

3. (also referred to in Table I as BAAB)

4. (also referred to in Table I as BAAA)

Disaccharides 1, 2, 3 and 4 can then be linked together in variouscombinations to provide oligosaccharides having a sequence withdifferent patterns for derivatization. To exemplify this concept, threedifferent decasaccharides are described below that are differentcombinations of the four disaccharides. The disaccharides are linkedtogether by deprotecting the levulinoyl at position R3 and the benzoylat position R4, and coupling the disaccharide structures to one anotherusing TMSOTf-coupling as described in Lohman et. al. J. Org. Chem. 2004,69(12), 4081-4093.

The following structure (i) is a combination of the followingdisaccharides: 1-2-3-4-1:

Structure (ii) provided below is a combination of the followingdisaccharides 2-3-4-1-2:

Structure (iii) provided below is a combination of the followingdisaccharides 1-3-2-4-3:

Such methods can be used to provide a collection or library of protectedoligosaccharides, e.g., having any combination of disaccharidesdescribed herein, e.g., any combination of disaccharides 1-16 of Table Ior of disaccharides 1-16 of Table II. The oligosaccharide can then bedeprotected at a class of protected positions, e.g., positions having aprotecting group that allows derivatization, e.g., sulfation, to providean oligosaccharide with unprotected moieties and forming substituents,e.g., derivative moieties, e.g., sulfate moieties. An oligosaccharidehaving a class of deprotected moieties at various positions in theoligosaccharide can be, e.g., sulfated at those positions using knowntechniques. For example, the oligosaccharide can be treated with sulfurtrioxide-pyridine complex (e.g., with pyridine as solvent) to sulfatethe deprotected moieties. For example, protected decasaccharides ofstructures i, ii and iii, provided above, can be treated withsulfur-trioxide-pyridine complex in the presence of pyridine, to providethe following derivatized oligosaccharides. Example 7 depicts such aprocedure being followed on a disaccharide as an experimental proof ofconcept.

Other exemplary methods of providing an oligosaccharide having apreselected sequence are described in FIGS. 8 to 11. Oligosaccharidesdescribed in these Figures are embodiments of the disclosure.

EXAMPLES Example 1 Synthesis of Glucosamine Synthon

The synthesis of the Glucosamine synthon is depicted in FIG. 12.

Synthesis of Compound 30 of FIG. 12

Compound 30 was obtained from Glucosamine hydrochloride as a white solidas described by Orgueira, H. A. et. al. Chem. Eur. J. 2003, 9 (1),140-69. The product was characterized by ¹H NMR.

Synthesis of Compound 31 of FIG. 12

Compound 30 (14 g, 0.0314 mol) was dissolved in methanol (175 ml) andsodium methoxide (25% in MeOH, 2.1 ml) was added and reaction stirredfor 15 mins. After 15 mins, Dowex-50 acidic resin was added to the aboveuntil the reaction mixture pH reached 6. The Dowex resin wassubsequently filtered off and the solvent was removed under vacuum toafford a brown colored reaction mass. The crude compound was purified bycolumn chromatography to obtain a yellow viscous oil as product (10 g,100% yield).

This yellow viscous oil (10 g, 0.0313 mol) was co-evaporated withtoluene and finally dissolved in dry acetonitrile (110 ml). p-Toluenesulphonic acid monohydrate (0.18 g, 0.00094 mol) and anisaldehydedimethyl acetal (11.42 g, 0.0627 mol) were added and the reaction massallowed to stir overnight at room temperature. On completion, triethylamine (1.35 ml) was added and the solvent was removed under vacuum.Purification was carried out by flash chromatography on silica gel using5% ethyl acetate as an eluant to afford pale yellow viscous oil as aproduct (8.8 g, 65% yield). The product was characterized by ¹H NMR.

Synthesis of Compound 52 of FIG. 12

Compound 31(27 g, 0.0619 mol) was dissolved in DCM (240 ml), to which 4A° molecular sieves (43 g) and benzyl bromide (53.45 g, 0.30 mol) wereadded and the reaction allowed to stir for 30 mins at room temperature.Silver (I) oxide (41.5 g, 0.179 mol) was added and the reaction wasstirred for 18 hrs in the dark. The silver (I) oxide was then filteredoff through celite bed and the filtrate concentrated under vacuum.Isolation of the product was done by column chromatography using 5%ethyl acetate as an eluant to get white solid (20 g, 61.5% yield). Theproduct was characterized by ¹H NMR.

Synthesis of Compound 53 of FIG. 12

A mixture of Compound 52 (20 g, 0.037 mol) and sodium cyanoborohydride(11.87 g, 0.189 mol) in anhydrous DMF (270 ml) containing 4 A° molecularsieves (9.6 g) was cooled to 0° C. under vigorous stirring. A solutionof trifluoroacetic acid (26 ml) in anhydrous DMF (146 ml) was added andreaction stirred for additional 2 h at 0° C. After 2 h the reaction masswas allowed to stir at room temperature for 18 h. On completion,reaction was quenched with triethylamine and then filtered andconcentrated under vacuum. The residue was dissolved in dichloromethaneand then washed with saturated sodium bicarbonate, dried over anhydroussodium sulphate and concentrated in vacuum, before purification bypreparative HPLC. Yield: 8 g (40%). The product was characterized by ¹HNMR.

Example 2 Synthesis of Iduronic Acid Synthons

The synthesis of the Iduronic acid synthons is depicted in FIG. 13.

Synthesis of Compound 6 of FIG. 13

Compound 6 was obtained from diacetone glucose (Compound I) through aseries of chemical transformations as detailed in Lohman, G. J. S. et.al. J. Org. Chem. 2003, 68 (19), 7559-7561.

Synthesis of Compound 7 of FIG. 13

Compound 6 (14.0 g) was dissolved in DCM (100 ml) and cooled to 0° C. Tothis was added levulinic acid (7.7 g), DIPC (9.7 ml) and DMAP (8.1 g) at0° C. and light was excluded. The reaction was stirred overnight at roomtemperature. On completion, the reaction mixture was diluted withEtOAc:Hexane (1:1, 200 ml), passed through a silica plug, concentratedand subjected to flash column chromatography using hexane: EtOAc systemto yield yellow oil (12.5 g). The product was characterized by ¹H NMRand LC/MS.

The yellow oil from previous step (12.5 g) was dissolved intrifluoroacetic acid (90% aqueous, 100 ml) and was stirred for 1 hr. Thesolvent was removed under vacuum and by co-evaporation with toluene.After removal of TFA, the reaction mixture was dissolved in DCM (50 ml),and Imidazole (3.9 gm) and tert-butyl dimethyl silyl chloride (4.75 g)were added. The reaction mixture was stirred overnight at roomtemperature. The reaction mixture was then diluted with ethyl acetate,washed with water, 1N HCl and water, dried, concentrated and subjectedto flash column chromatography using Hexane: EtOAc solvent system toyield Compound 7 (9.1 g). The product was characterized by ¹H NMR andLC/MS.

Synthesis of Compound 8 of FIG. 13

Compound 7 (5.0 g) was dissolved in DCM (10 ml) and cooled to 0° C. andto this was added pyridine (2.0 ml) at 0° C. under nitrogen. Theresultant solution was stirred for 10 minutes and benzoyl chloride (1.5ml) was added. The reaction mixture was then stirred for 48 hrs (at 7-8°C.) under nitrogen. The reaction mixture was then diluted with DCM (50ml), washed with water, 1N.HCl, 10% sodium bicarbonate and brinesolution. This was dried, concentrated and subjected to flash columnchromatography using Hexane: EtOAc solvent system to yield pure Compound8 (3.7 g). The product was characterized by ¹H NMR and LC/MS.

Synthesis of Compound 25 of FIG. 13

Compound 6 (12.0 gm) was dissolved in DCM (35 ml) and cooled to 0° C.and to this was added pyridine (7.2 ml). The solution was then stirredfor 10 minutes following which benzoyl chloride (5.5 ml) was added. Thisreaction mixture was then stirred for 48 hrs (at 7-8° C.) undernitrogen. On completion, the reaction mixture was diluted with DCM (50ml), washed with water, 1N HCl, 10% sodium bicarbonate and brinesolution. The organic layers was dried, concentrated and subjected toflash column chromatography using Hexane: EtOAc solvent system to yieldcompound (10.0 gm). The product was characterized by ¹H NMR and LC/MS.

Compound from previous step (9.0 gm) was dissolved in trifluoroaceticacid (90% aqueous, 72 ml)) and stirred for 1 hour. The solvent wasremoved under vacuum by co-evaporation with toluene. The resultant oilwas dissolved in DCM (50 ml), and imidazole (3.1 gm) and tert-butyldimethyl silyl chloride (3.75 gm) added. The reaction mixture wasstirred overnight at room temperature. On completion, the reactionmixture was diluted with ethyl acetate, washed with water, 1N HCl andwater, dried, concentrated and subjected to flash column chromatographyusing Hexane: EtOAc solvent system to yield Compound 25 (6.0 gm). Theproduct was characterized by ¹H NMR and LC/MS.

Synthesis of Compound 26 of FIG. 13

Compound 25 (6.0 gm) was dissolved in DCM (100 ml) and cooled to 0° C.To this was added Levulinic acid (1.9 ml), DIPC (2.7 ml) and DMAP (2.3gm) at 0° C. and reaction stirred overnight at room temperature underlight exclusion. The reaction mixture was diluted with EtOAc:Hexane(1:1, 100 ml) and passed through a silica plug. It was then concentratedand subjected to column chromatography using Hexane:EtOAc system toyield pure Compound 26 (4.0 gm). The product was characterized by ¹H NMRand LC/MS.

Example 3 Synthesis of Disaccharide 54 of FIG. 14

The synthesis of Disaccharide 54 is depicted in FIG. 14.

1. Generation of Disaccharide Donor 27 of FIG. 14

Compound 26 (0.62 g, 0.1 mmol) was dissolved in freshly distilled THF(10 mL) and cooled to 0° C. To this was added, glacial acetic acid (110μL, 2.0 mmol) followed by TBAF (1.0M in THF, 2.0 mL). The reactionmixture was stirred for 30 min at 0° C. and then diluted with 200 mL ofethyl acetate. The organic layer was washed with NH₄Cl, NaHCO₃, brine,and then water, dried over anhydrous Na₂SO₄, filtered and concentratedto yield a yellow oil (461 mg) which was used without furtherpurification in the next step.

The crude oil obtained was co-evaporated with toluene under high vacuumand dissolved in dry dichloromethane (10 mL). The solution was cooled to0° C. and trichloroacetonitrile (1.0 mL, 10 mmol) and DBU (14 μL, 0.1mmol) were added. The reaction mixture was stirred for 20 min at 0° C.and then poured onto a flash chromatography column on silica gel toobtain pure Compound 27 (415 mg, 63% yield). The compound wascharacterized by ¹H NMR.

2. Synthesis of Disaccharide 54 of FIG. 14

The trichloroacetimidate (Compound 27, 415 mg, 0.64 mmol) and theCompound 53 (320 mg, 0.58 mmol) were combined and dissolved in drydichloromethane (10 mL). 4 A molecular sieves (0.5 g) was added and themixture was stirred for 15 min at room temperature. Triethylsilyltrifluoromethanesulfonate (65 μL, 0.25 mmol) was added at roomtemperature. After 30 min, the reaction was quenched with triethylamine,concentrated under reduced pressure and the residue was purified byflash chromatography on silica gel to give a ‘α and β’ disaccharidemixture (408 mg, α:β=6:1). Further purification by column chromatographyyielded pure a Disaccharide 54 (310 mg). The compound was characterizedby ¹H NMR, ¹³C NMR, 2D NMR (HSQC, COSY, TOCSY) and ESI-MS.

Example 4 Synthesis of Disaccharide 55 of FIG. 15

The synthesis of Disaccharide 55 is depicted in FIG. 15.

1. Synthesis of Disaccharide Donor 9 of FIG. 15

Compound 8 (1.2 g) was dissolved in freshly distilled THF (10 mL) andcooled to 0° C. To this was added glacial acetic acid (110 μL, 2.0 mmol)followed by THF (1.0 M of TBAF, 1.94 mL). The reaction mixture wasstirred for 15 min and then diluted with 200 mL of ethyl acetate. Theorganic layer was washed with NH₄Cl, NaHCO₃, brine, and water and thendried over anhydrous Na₂SO₄, filtered and then concentrated to yield alight yellow oil (560 mg). This compound was used without furtherpurification in the next step. Crude compound was dissolved in drydichloromethane (8 mL). The solution was cooled to 0° C., andtrichloroacetonitrile (1.0 mL, 10 mmol) and DBU (14 μL, 0.1 mmol) wereadded. The reaction mixture was stirred for 20 min and then poured ontoflash chromatography column on silica gel to yield Compound 9 (320 mg,51% yield). The compound was characterized by ¹H NMR.

2. Synthesis of Disaccharide 55 of FIG. 15

Trichloroacetimidate 9 (320 mg, 0.5 mmol) and compound 53 (240 mg, 0.45mmol) were combined and dissolved in anhydrous dichloromethane (10 mL).4 Å Molecular sieves (0.5 g) and triethylsilyl trifluoromethanesulfonate(40 μL, 0.2 mmol) was added and the mixture was stirred for 15 min atroom temperature. On completion, the reaction was quenched withtriethylamine, concentrated under reduced pressure and the residue waspurified by flash chromatography on silica gel to give an ‘α and β’disaccharide mixture (300 mg total, α:β=5:1). Further purification bycolumn chromatography afforded pure a form of Disaccharide 55 (210 mg).The compound was characterized by ¹H NMR, ¹³C NMR, 2D NMR (HSQC, COSY,TOCSY) and ESI-MS.

Example 5 Synthesis of Tetrasaccharide 59 of FIG. 16

The synthesis of Tetrasaccharide 59 is depicted in FIG. 16.

1. Synthesis of Disaccharide Acceptor 56 of FIG. 15

Disaccharide 55 (0.41 g, 0.4 mmol) was dissolved in 5 mL of MeOH and 5mL of 1% NaOH in MeOH was added at 0° C. and the reaction mixturesubsequently warmed to room temperature. The reaction mixture wasstirred for 1 hour and acetic acid (AcOH) was added to quench thereaction. The reaction mixture was concentrated and the residue waspurified by flash column chromatography to yield 235 mg of acceptor 56as a white solid (72% yield). The compound was characterized by ¹H NMR.

2. Synthesis of Disaccharide Donor 57 of FIG. 15

Disaccharide 54 (710 mg, 0.7 mmol) was dissolved in freshly distilledTHF (15 mL), solution cooled to 0° C. and acetic acid (92 μL, 1.5 mmol)was added, followed by TBAF (1.0 M in THF, 1.4 mL). The reaction mixturewas kept stiffing for 20 min and diluted with 100 mL of ethyl acetate.The organic layer was washed with NH₄Cl, NaHCO₃, brine, and water andthen dried over anhydrous Na₂SO₄. The sample was filtered andconcentrated to yield a yellow oil (610 mg).

The crude oil thus obtained, was dissolved in anhydrous dichloromethane(15 mL). The solution was then cooled to 0° C., andtrichloroacetonitrile (0.7 mL, 7 mmol) and DBU (10 μL, 0.07 mmol) wereadded. The reaction mixture was stirred for 20 min and then poured ontoa flash chromatography column on silica gel to purify disaccharide 57(502 mg, 68% yield). The compound was characterized by ¹H NMR.

3. Assembly of Tetrasaccharide 59 of FIG. 16

Trichloroacetimidate 57 (302 mg, 0.29 mmol) and disaccharide acceptor 56(235 mg, 0.29 mmol) were combined and dissolved in anhydrous toluene (15mL). 4° A molecular sieves (1 g) and triethylsilyltrifluoromethanesulfonate (26 μL, 0.12 mmol) was added at −60° C. Thereaction mixture was stirred for one hour. The reaction was thenquenched with triethylamine, filtered and concentrated under reducedpressure. The residue was purified by flash chromatography on silica gelto give α- and β-tetrasaccharides. Further purification (flashchromatography on silica gel) afforded the pure α form oftetrasaccharide 58 (65 mg, 13% yield). The compound was characterized by¹H NMR, ¹³C NMR, 2D NMR (HSQC, COSY, TOCSY) and ESI-MS.

Tetrasaccharide 58 (65 mg, 0.038 mmol) was dissolved in a mixture of 2mL dry CH₂Cl₂ and 0.5 mL of anhydrous pyridine. To this was added, Lev₂O(39 mg, 0.19 mmol) and a small amount of DMAP at room temperature. After30 min, the reaction mixture was diluted with 50 mL of CH₂Cl₂ and theorganic layer was washed with cold NaHCO₃, 1% HCl, and water. Afterdrying over anhydrous Na₂SO₄, the solution was filtered andconcentrated. The remaining residue was purified by flash chromatographyto yield the desired final tetrasaccharide 59 (60 mg, 88% yield). Thecompound was characterized by ¹H NMR, ¹³C NMR, 2D NMR (HSQC, COSY,TOCSY) and ESI-MS.

Example 6 Synthesis of Hexasaccharide 62 of FIG. 17

The synthesis of Hexasaccharide 60 is depicted in FIG. 17.

1. Synthesis of Tetrasaccharide Acceptor 60 of FIG. 17

Tetrasaccharide 58 (0.18 g, 0.11 mmol) was dissolved in 5 mL of MeOH. 5mL of 1% NaOH in MeOH was added at 0° C. and the reaction mixture wassubsequently stirred at room temperature for another 4 hours after whichtime the reaction was quenched by addition of AcOH. The reaction mixturewas concentrated and the residue was purified by flash columnchromatography to give 68 mg of white solid (Tetrasaccharide 60, 43%yield). The compound was characterized by ¹H NMR.

2. Assembly of Hexasaccharide 62 of FIG. 17

Trichloroacetimidate disaccharide 57, (48 mg, 0.046 mmol) andtetrasaccharide acceptor 60 (68 mg, 0.045 mmol) were combined anddissolved in dry toluene (8 mL). 4° A Molecule sieves (100 mg) andtriethylsilyl trifluoromethanesulfonate (4.1 μL, 0.011 mmol) was addedat −60° C. and the mixture was stirred for one hour. The reaction wasthen quenched with triethylamine, the mixture filtered and thenconcentrated under reduced pressure. The residue was purified by flashchromatography on silica gel to give a mixture of cc and f3hexasaccharides and further purification by flash chromatographyafforded the pure cc form hexasaccharide 61 (10 mg, 9% yield). Thecompound was characterized by ¹H NMR, ¹³C NMR, 2D NMR (HSQC, COSY,TOCSY) and ESI-MS.

Hexasaccharide 61 (10 mg, 0.004 mmol) was dissolved in a mixture of 2 mLof dry CH₂Cl₂ and 0.5 mL of anhydrous pyridine. To this was added Lev₂O(4 mg, 0.02 mmol) and a small amount of DMAP at room temperature. After30 min, the reaction mixture was diluted with 20 mL of CH₂Cl₂ and theorganic layer was washed with cold NaHCO₃, 1% HCl and water. Afterdrying over anhydrous Na₂SO₄, the mixture was filtered, concentrated andthe residue was then purified by column chromatography to yield thedesired final hexasaccharide, 62 (8 mg, 75% yield). The compound wascharacterized by ¹H NMR, ¹³C NMR, 2D NMR (HSQC, COSY, TOCSY) and ESI-MS.

Example 7 Synthesis of Monosulfated Disaccharide 63

The synthesis of the monosulfated Disaccharide 63 is shown in FIG. 18.

Disaccharide 54 (0.1 g, 0.099 mmol) was dissolved in 8 mL ofEtOH/Toluene (2/1 v/v) and hydrazine acetate (91 mg, 0.99 mmol) wasadded at room temperature. The reaction mixture was stirred for 2 hoursand then diluted with ethyl acetate (20 mL). The organic layer waswashed with water, dried with NaSO₄, filtered, and concentrated viaevaporation. The resulting residue was purified by column chromatographyon silica gel to provide 88 mg (98% crude yield) of C-20H compound to beused in the next step.

Crude compound from previous step (44 mg, 0.048 mmol) was dissolved in 5mL of pyridine and sulfur-trioxide pyridine complex (SO₃Py, 23 mg, 0.144mmol) was added.

The reaction mixture was left to stir at 45° C. for 3 hours after whichMeOH was added to quench the reaction. The mixture was then concentratedand the resulting residue was purified by column chromatography onsilica gel to give the C-2 O-sulphated compound as a ‘pyridine’ salt.Dried pyridinium salt was dissolved in 5 mL of MeOH and then 1 mL ofNaHCO₃ (10% aqueous) was added. The reaction mixture was thenconcentrated to give crude sodium salt product which was purified byflash chromatography to yield the final product as the ‘sulfated sodiumsalt’ (Compound 63, 45 mg, 94% yield).

What is claimed:
 1. An oligosaccharide comprising disaccharide units, wherein the oligosaccharide is a tetrasaccharide, a hexasaccharide, an octasaccharide, a decasaccharide or a dodecasaccharide, and wherein the identity of each disaccharide unit is independent of the identity of the other disaccharide units within the oligosaccharide, the oligosaccharide comprising the formula:

wherein each tetrasaccharide comprises X1, each hexasaccharide comprises X1 and X2, each octasaccharide comprises X1, X2, and X3, each decasaccharide comprises X1, X2, X3, and X4 and each dodecasaccharide comprises X1, X2, X3, and X4; and wherein each of X1, X2, X3 and X4 is independently A or B, and wherein A is

wherein R₈ for each occurrence of A or B is independently a hydrogen or an alkyl group; R₅ for each occurrence of A or B is an amine containing protecting group that, independently for each occurrence, allows for either sulfation or acetylation; and wherein R₁, R₂, R₆ and R₇ for each occurrence of A or B is independently a protecting group selected from a first protecting group that allows for sulfation and a second protecting group that does not allow for sulfation, and wherein in at least one occurrence of A or B, at least one of R₁, R₂, R₆, and R₇ is the first protecting group and at least one is the second protecting group.
 2. The oligosaccharide of claim 1, having a preselected pattern of protecting groups, which when sulfated will provide a preselected pattern of sulfation.
 3. The oligosaccharide of claim 1, wherein if more than one position of R₁, R₂, R₆ and R₇ in the oligosaccharide is a protecting group that allows sulfation, the protecting group that allows sulfation is the same protecting group at each position to be sulfated.
 4. The oligosaccharide of claim 1, wherein if more than one position of R₁, R₂, R₆, and R₇ in the oligosaccharide is a protecting group that does not allow sulfation, the protecting group that does not allow sulfation is the same protecting group at each position that will not be sulfated.
 5. The oligosaccharide of claim 1, wherein if more than one position of R₅ in the oligosaccharide is a protecting group that allows sulfation, the protecting group that allows for sulfation is the same protecting group at each R₅ position to be sulfated.
 6. The oligosaccharide of claim 1, wherein if more than one position of R₅ is a protecting group that allows acetylation, the protecting group that allows acetylation is the same protecting group at each R₅ position to be acetylated.
 7. A method of making an oligosaccharide comprising disaccharide units, wherein the oligosaccharide is a tetrasaccharide, a hexasaccharide, an octasaccharide, a decasaccharide or a dodecasaccharide of a preselected pattern of derivatization, wherein the identity of each disaccharide unit is independent of the identity of the other disaccharide units within the oligosaccharide, and wherein the oligosaccharide comprises:

wherein each tetrasaccharide comprises X1, each hexasaccharide comprises X1 and X2, each octasaccharide comprises X1, X2, and X3, each decasaccharide comprises X1, X2, X3, and X4 and each dodecasaccharide comprises X1, X2, X3, and X4; and wherein each of X1, X2, X3 and X4 is independently A or B, and wherein

wherein R₈ for each occurrence of A or B is independently a hydrogen or an alkyl group, and wherein R₅ for each occurrence of A or B is an amine containing protecting group that, independently for each occurrence, either allows for derivatization or does not allow for derivatization, and wherein R₁, R₂, R₆ and R₇ for each occurrence of A or B is a protecting group selected from either a first protecting group that allows derivatization or a second protecting group that does not allow derivatization, the method comprising: providing a first protected saccharide structure, wherein the saccharide is a monosaccharide of the following structure:

wherein R₁, R₂, R₃, R₈ and R₉ are protecting groups; R₁ and R₂ are independently a protecting group selected from a first protecting group that allows derivatization and a second protecting group that does not allow derivatization; R₈ for each occurrence of A or B is independently a hydrogen or an alkyl group; and R₃ and R₉ are not the same protecting group as any of R₁, R₂ and R₈; providing a second saccharide structure, wherein the saccharide is a monosaccharide of the following structure:

wherein R₄, R₅, R₆, R₇, and R₁₀ are protecting groups; R₆ and R₇ are independently a protecting group selected from a first protecting group that allows derivatization and a second protecting group that does not allow derivatization; R₅ for each is an amine containing protecting group; and R₄ and R₁₀ are not the same protecting group as any of R₅, R₆ and R₇; and attaching the first saccharide structure to the second saccharide structure, to thereby make an oligosaccharide of the preselected pattern of derivatization.
 8. The method of claim 7, wherein if more than one of R₁ and R₂ in the first saccharide moiety is a protecting group that allows derivatization, the protecting group that allows derivatization is the same protecting group at each of R₁ , and R₂.
 9. The method of claim 7, wherein if more than one of R₁ and R₂ in the first saccharide moiety is a protecting group that does not allow derivatization, the protecting group that does not allow derivatization is the same protecting group at each of R₁ and R₂.
 10. The method of claim 7, wherein the oligosaccharide having a preselected pattern of derivatization is a hexasaccharide, an octasaccharide, a decasaccharide, or a dodecasaccharide.
 11. The method of claim 7, further comprising deprotecting the protecting group amendable to derivatization to form an unprotected moiety.
 12. The method of claim 11, further comprising forming derivative moieties at the deprotected position or positions.
 13. The method of claim 7, wherein the second saccharide structure includes N-acetylglucosamine.
 14. The method of claim 7, wherein the first saccharide structure includes a iduronic acid.
 15. An oligosaccharide made by the method of claim
 12. 16. The oligosaccharide of claim 1, wherein the alkyl group at R₈ for each occurrence of A or B is independently selected from a methyl, ethyl, propyl, butyl, and pentyl group.
 17. The oligosaccharide of claim 1, wherein the selection of one or more of R₁, R₂, R₅, R₆, and R₇ can differ between a first and second group A, an A and B, or a first and second B.
 18. The oligosaccharide of claim 1, wherein the protecting group that allows sulfation is a benzyl and/or a benzyl containing group and the protecting group that does not allow sulfation is a benzoyl and/or a benzoyl containing group.
 19. The oligosaccharide of claim 1, wherein the protecting group that allows sulfation is a benzoyl and/or a benzoyl containing group and the protecting group that does not allow sulfation is a benzyl and/or a benzyl containing group.
 20. The oligosaccharide of claim 1, wherein the amine containing protecting group that allows sulfation is a carbamate or substituted carbamate group and the amine containing protecting that does not allow sulfation is an azide or NHCBz or NHTroc or NHFmoc.
 21. The oligosaccharide of claim 1, wherein the amine containing protecting group that allows sulfation is an azide or NHCBz or NHTroc or NHFmoc and the amine containing protecting group that does not allow sulfation is a carbamate or substituted carbamate group.
 22. The oligosaccharide of claim 1, wherein R₁ at each occurrence is a benzoyl or benzyl group; R₂ at each occurrence is a benzyl group; R₅ at each occurrence is an N₃ group or an NHCBz group; and R₆ at each occurrence is a benzoyl or benzyl group; and R₇ is a benzoyl or benzyl group.
 23. The oligosaccharide of claim 1, wherein R₁, R₂, R₆, and R₇ for each occurrence of A or B each of R₁, R₂, R₆, and R₇ are distinct from one another or a subset of R₁, R₂, R₆, and R₇ are distinct from one another.
 24. The oligosaccharide of claim 1, wherein the amine protecting group at R₅ for each occurrence of A or B is independently a carbamate or a substituted carbamate group.
 25. An oligosaccharide comprising disaccharide units, wherein the oligosaccharide is a tetrasaccharide, a hexasaccharide, an octasaccharide, a decasaccharide or a dodecasaccharide and the identity of each disaccharide unit is independent of the identity of the other disaccharide units within the oligosaccharide, wherein the oligosaccharide comprising the formula:

wherein each tetrasaccharide comprises X5, each hexasaccharide comprises X5 and X6, each octasaccharide comprises X5, X6, and X7, each decasaccharide comprises X5, X6, X7, and X8 and each dodecasaccharide comprises X5, X6, X7, and X8; wherein each of X5, X6, X7 and X8 is independently C or D, and wherein

and wherein R₈ for each occurrence of C or D is a hydrogen or an alkyl group; R₅, for each occurrence of C or D is an amine containing protecting group that, independently for each occurrence, allows either sulfation or acetylation; and wherein R₁, R₂, R₆, and R₇ for each occurrence of C or D is independently a protecting group selected from either a first protecting group that allow for sulfation, or a second protecting group that does not allow for sulfation.
 26. The oligosaccharide of claim 25, having a preselected pattern of protecting groups, which when sulfated and/or acetylated will provide a preselected pattern of sulfation or acetylation.
 27. The oligosaccharide of claim 25, wherein if more than one position of R₁, R₂, R₆, and R₇ in the oligosaccharide is a protecting group that allows sulfation, the protecting group that allows sulfation is the same protecting group at each of R₁, R₂, R₆, and R₇.
 28. The oligosaccharide of claim 25, wherein if more than one position of R₁, R₂, R₆, and R₇ in the oligosaccharide is a protecting group that does not allow sulfation, and the protecting group that does not allow sulfation is the same protecting group at each of R₁, R₂, R₆, and R₇.
 29. The oligosaccharide of claim 25, wherein if more than one position of R₅ in the oligosaccharide is a protecting group that allows sulfation, the protecting group that allows for sulfation is the same protecting group at each R₅ position to be sulfated.
 30. The oligosaccharide of claim 25, wherein if more than one position of R₅ is a protecting group that allows acetylation, the protecting group that allows acetylation is the same protecting group at each R₅ position to be acetylated.
 31. The oligosaccharide of claim 25, wherein the alkyl group R₈ for each occurrence of C or D is independently selected from a methyl, ethyl, propyl, butyl, or pentyl group.
 32. The oligosaccharide of claim 25, wherein the selection of one or more of R₁, R₂, R₅, R₆, and R₇ can differ between a first and second group C, a C and D, or a first and second D.
 33. The oligosaccharide of claim 25, wherein the protecting group that allows sulfation is a benzyl and/or a benzyl containing group and the protecting group that does not allow sulfation is a benzoyl and/or a benzoyl containing group.
 34. The oligosaccharide of claim 25, wherein the protecting group that allows sulfation is a benzoyl and/or a benzoyl containing group and the protecting group that does not allow sulfation is a benzyl and/or a benzyl containing group.
 35. The oligosaccharide of claim 25, wherein the amine containing protecting group that allows sulfation is a carbamate or a substituted carbamate group and the amine containing protecting that does not allow sulfation is an azide or NHCBz or NHTroc or NHFmoc.
 36. The oligosaccharide of claim 25, wherein the amine containing protecting group that allows sulfation is an azide or NHCBz or NHTroc or NHFmoc and the amine containing protecting group that does not allow sulfation is a carbamate or a substituted carbamate group.
 37. The oligosaccharide of claim 25, wherein the protecting group of any of R₁, R₂, R₆, and R₇ for each occurrence of C or D is independently a hydroxyl protecting group.
 38. The oligosaccharide of claim 37, wherein the hydroxyl protecting group, is a silyl ether, ethyl ether, substituted benzyl ether or ester group.
 39. The oligosaccharide of claim 25, wherein the amine protecting group at R₅ for each occurrence of C or D is independently a carbamate or a substituted carbamate group.
 40. The oligosaccharide of claim 25, wherein the protecting group at of any of R₁, R₂, R₆, and R₇ that allows sulfation is selected from levulinoyl, benzyl (Bn), benzoyl (Bz), methoxybenzyl (MPM), azide, allyl and silyl ether protecting group, and the protecting group that does not allow sulfation is selected from levulinoyl, benzyl, benzoyl, MPM, azide, allyl and silyl ether protecting group, wherein the protecting group that allows sulfation and the protecting group that does not allow sulfation are orthogonal protecting groups.
 41. The oligosaccharide of claim 25, wherein the protecting group that allows sulfation is a levulinoyl and the protecting group that does not allow sulfation is a benzoyl and/or a benzoyl containing group.
 42. The oligosaccharide of claim 25, wherein the protecting group that allows sulfation is a benzoyl and/or a benzoyl containing group and the protecting group that does not allow sulfation is levulinoyl.
 43. The oligosaccharide of claim 25, wherein the amine containing protecting group that allows sulfation is an azide and the amine containing protecting group that does not allow sulfation is a carbamate or substituted carbamate group.
 44. The oligosaccharide of claim 25, wherein the amine containing protecting group that allows derivatization is a a carbamate or substituted carbamate group and the amine containing protecting group that does not allow derivatization is an azide.
 45. The oligosaccharide of claim 25, wherein R₁ at each occurrence is independently a benzoyl or benzyl containing group; R₂ at each occurrence is independently a benzyl group; R₅ at each occurrence is independently an N₃ group or an NHCBz group; and R₆ at each occurrence is independently a benzoyl or containing benzyl group; and R₇ at each occurrence is independently a benzoyl or benzyl containing group.
 46. The oligosaccharide of claim 25, wherein R₁, R₂, R₆, and R₇ for each occurrence of C or D, each of R₁, R₂, R₅, R₆, and R₇ are distinct from one another or a subset of R₁, R₂, R₆, and R₇ are distinct from one another.
 47. The method of claim 7, wherein the alkyl group is a methyl, ethyl, propyl, butyl, or pentyl group.
 48. The method of claim 7, wherein the selection of one or more of R₁, R₂, R₆, and R₇ can differ between a first and second group A, an A and B, or a first and second B.
 49. The method of claim 7, wherein R₁, R₂, R₆ and R₇ are protecting groups selected from either a first protecting group that allows derivatization, or a second protecting group that does not allow derivatization, and wherein the identity of the protecting group at any of R₁, R₂, R₅, R₆ and R₇ is independent of the identity of a protecting group at any of the other positions; and wherein R₈ is a hydrogen or an alkyl group.
 50. The method of claim 7, wherein the alkyl group is a methyl, ethyl, propyl, butyl, or pentyl group.
 51. The method of claim 7, wherein the protecting group that allows derivatization is a benzyl and/or a benzyl containing group and the protecting group that does not allow derivatization is a benzoyl and/or a benzoyl containing group.
 52. The method of claim 7, wherein the protecting group that allows derivatization is a benzoyl and/or a benzoyl containing group and the protecting group that does not allow derivatization is a benzyl and/or a benzyl containing group.
 53. The method of claim 7, wherein the amine containing protecting group that allows sulfation is a carbamate or substituted carbamate group and the amine containing protecting that does not allow sulfation is an azide.
 54. The method of claim 7, wherein the amine containing protecting group that allows sulfation is an azide and the amine containing protecting group that does not allow sulfation is a carbamate or substituted carbamate group.
 55. The method of claim 7, wherein R₁ at each occurrence is independently a benzoyl or benzyl group; R₂ at each occurrence is independently a benzyl group; and R₆ at each occurrence is independently is a benzoyl or benzyl group; and R₇ at each occurrence is independently is a benzoyl or benzyl group.
 56. The method of claim 7, wherein R₁, R₂, R₆, and R₇ for each occurrence of A or B, each of R₁, R₂, R₆, and R₇ are distinct from one another or a subset of R₁, R₂, R₆, and R₇ are distinct from one another.
 57. The method of claim 7, wherein the protecting group that allows derivatization is sulfation or acetylation and the protecting group that does not allow derivatization is sulfation or acetylation.
 58. A method of making an oligosaccharide comprising disaccharide units, wherein the oligosaccharide is a tetrasaccharide, a hexasaccharide, an octasaccharide, a decasaccharide or a dodecasaccharide of a preselected pattern of derivatization, wherein the identity of each disaccharide unit is independent of the identity of the other disaccharide units within the oligosaccharide, and wherein the oligosaccharide comprises:

wherein each tetrasaccharide comprises X5, each hexasaccharide comprises X5 and X6, each octasaccharide comprises X5, X6, and X7, each decasaccharide comprises X5, X6, X7, and X8 and each dodecasaccharide comprises X5, X6, X7, and X8; wherein each of X5, X6, X7 and X8 is independently C or D, and wherein

and wherein R₈ for each occurrence of C or D is independently a hydrogen or an alkyl group, and wherein R₅ for each occurrence of C or D is an amine containing protecting group that, independently for each occurrence, either allows for derivatization or does not allow for derivatization, and wherein R₁, R₂, R₆, and R₇ for each occurrence of C or D is a protecting group selected from either a first protecting group that allows derivatization or a second protecting group that does not allow derivatization, the method comprising: providing a first protected saccharide structure, wherein the saccharide is a monosaccharide of the following structure:

wherein R₄, R₅, R₆, R₇, and R₁₀ are protecting groups; R₆, and R₇ are independently a protecting group selected from a first protecting group that allows derivatization and a second protecting group that does not allow derivatization; R₅ for each is an amine containing protecting group; and R₄, and R₁₀ are not the same protecting group as any of R₅, R₆ and R_(7;) providing a second saccharide structure, wherein the saccharide is a monosaccharide of the following structure:

wherein R₁, R₂, R₃, R₈, and R₉ are protecting groups; R_(l) and R₂ are independently a protecting group selected from a first protecting group that allows derivatization and a second protecting group that does not allow derivatization; R₈ for each occurrence of A or B is independently a hydrogen or an alkyl group; and R₃, and R₉ are not the same protecting group as any of R₁, R₂ and R₈; and attaching the first saccharide structure to the second saccharide structure, to thereby make an oligosaccharide of the preselected pattern of derivatization.
 59. The method of claim 58, wherein if more than one position of R₆, and R₇ in the first saccharide moiety is a protecting group that allows derivatization, the protecting group that allows derivatization is the same protecting group at each of R₆, and R₇.
 60. The method of claim 58, wherein if more than one position of R₆, and R₇ in the first saccharide moiety is a protecting group that does not allow derivatization, the protecting group that does not allow derivatization is the same protecting group at each of R₆, and R₇.
 61. The method of claim 58, wherein the oligosaccharide having a preselected pattern of derivatization is a hexasaccharide, an octasaccharide, a decasaccharide, or a dodecasaccharide.
 62. The method of claim 58, further comprising deprotecting the protecting group amendable to derivatization to form an unprotected moiety.
 63. The method of claim 58, further comprising forming derivative moieties at the deprotected position or positions.
 64. The method of claim 58, wherein the alkyl group is a methyl, ethyl, propyl, butyl, or pentyl group.
 65. The method of claim 58, wherein the selection of one or more of R₁, R₂, R₆, and R₇ can differ between a first and second group C, a C and D, or a first and second D.
 66. The method of claim 58, wherein R₁, R₂, R₆ and R₇ are protecting groups selected from either a first protecting group that allows derivatization, or a second protecting group that does not allow derivatization, and wherein the identity of the protecting group at any of R₁, R₂, R₆ and R₇ is independent of the identity of a protecting group at any of the other positions.
 67. The method of claim 58, wherein the alkyl group is a methyl, ethyl, propyl, butyl, or pentyl group.
 68. The method of claim 58, wherein the protecting group that allows derivatization is a benzyl and/or a benzyl containing group and the protecting group that does not allow derivatization is a benzoyl and/or a benzoyl containing group.
 69. The method of claim 58, wherein the protecting group that allows derivatization is a benzoyl and/or a benzoyl containing group and the protecting group that does not allow derivatization is a benzyl and/or a benzyl containing group.
 70. The method of claim 58, wherein the amine containing protecting group that allows sulfation comprises a carbamate or substituted carbamate group and the amine containing protecting that does not allow sulfation comprises an azide.
 71. The method of claim 58, wherein the amine containing protecting group that allows sulfation comprises an azide and the amine containing protecting group that does not allow sulfation comprises a carbamate or substituted carbamate group.
 72. The method of claim 58, wherein the protecting group at any of R₁, R₂, R₆ and R₇ is a hydroxyl protecting group.
 73. The method of claim 72, wherein the hydroxyl protecting group, is a silyl ether, ethyl ether, substituted benzyl ether or ester group.
 74. The method of claim 58, wherein the amine protecting group is a carbamate or a substituted carbamate group.
 75. The method of claim 58, wherein the protecting group that allows derivatization is selected from levulinoyl, benzyl (Bn), benzoyl (Bz), methoxybenzyl (MPM), azide, allyl and silyl ether protecting group, and the protecting group that does not allow derivatization is selected from levulinoyl, benzyl, benzoyl, MPM, azide, allyl and silyl ether protecting group wherein the protecting group that allows derivatization and the protecting group that does not allow derivatization are orthogonal protecting groups.
 76. The method of claim 58, wherein the protecting group that allows derivatization is a benzoyl and/or a benzoyl containing group and the protecting group that does not allow derivatization is a benzyl and/or a benzyl containing group.
 77. The method of claim 58, wherein the protecting group that allows derivatization is a benzyl and/or a benzyl containing group and the protecting group that does not allow derivatization is a benzoyl and/or a benzoyl containing group.
 78. The method of claim 58, wherein the protecting group that allows derivatization is a levulinoyl and the protecting group that does not allow derivatization is a benzoyl and/or a benzoyl containing group.
 79. The method of claim 58, wherein the protecting group that allows derivatization is a benzoyl and/or a benzoyl containing group and the protecting group that does not allow derivatization is levulinoyl.
 80. The method of claim 58, wherein R₁ is a benzoyl or benzyl group; R₂ is a benzyl group; R₅ is an N₃ group or an NHCBz group; and R₆ is a benzoyl or benzyl group; and R₇ is a benzoyl or benzyl group.
 81. The method of claim 58, wherein R₁, R₂, R₅, R₆, and R₇ for each occurrence of C or D each of R₁, R₂, R₅, R₆, and R₇ are distinct from one another or a subset of R₁, R₂, R₅, R₆, and R₇ are distinct from one another.
 82. The method of claim 58, wherein the first saccharide structure includes an N-acetylglucosamine.
 83. The method of claim 58, wherein the second saccharide structure includes an iduronic acid.
 84. An oligosaccharide made by the method of claim
 58. 